Methods and compositions for treating cancer

ABSTRACT

Methods and compositions provided herein relate to the treatment of cancer. In some embodiments, the compositions have utility in the treatment of cancers including glioblastoma multiforme and lung cancer.

INCORPORATION BY REFERENCE TO RELATED APPLICATIONS

Any and all priority claims identified in the Application Data Sheet, orany correction thereto, are hereby incorporated by reference under 37CFR 1.57. This application is a continuation of U.S. application Ser.No. 15/030,713, filed Apr. 20, 2016, which is the national phase under35 U.S.C. §371 of PCT International Application No. PCT/US2014/061418,which has an International filing date of Oct. 20, 2014, which claimsthe benefit of U.S. Provisional Application No. 61/895,308 filed Oct.24, 2013. Each of the aforementioned applications is incorporated byreference herein in its entirety, and each is hereby expressly made apart of this specification.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under NIH Grant/ContractNumbers R01CA138212, R01CA133662, RC4CA156509 awarded by the NationalInstitutes of Health of the United States of America. The government hascertain rights in the invention.

FIELD OF THE INVENTION

Methods and compositions provided herein relate to the treatment ofcancer. In some embodiments, the compositions have utility in thetreatment of cancers including glioblastoma multiforme and lung cancer.

BACKGROUND OF THE INVENTION

The Central Brain Tumor Registry of the United States (CBTRUS) lists thetotal number of primary malignant brain tumor deaths for all 50 statesand the District of Columbia in 2012 is estimated to be 13,700.Glioblastomas (GBMs) are the most common brain malignancy with a mediansurvival of only 14.6 months in humans despite standard tri-modalitytreatment consisting of surgical resection, post-operative radiationtherapy and temozolomide chemotherapy. Therapy is almost never curativebecause the infiltrative nature of these tumors and their intrinsicresistance to radiation and chemotherapy. Even with optimal treatment,the median survival is less than 15 months, with only 10% of patientssurviving 2 years without disease recurrence. New therapeutic targetsare clearly needed to improve patient survival and quality of life forGlioblastomas and other cancers.

In addition, the Ewing's Sarcoma Family of Tumors (ESFT) are highlyaggressive tumors that occur in children, adolescents and young adultsin the bone and the soft tissues. They respond to chemotherapy, yet 75%to 80% of the patients who have developed metastatic ESFTs will die infive years despites high doses of chemotherapy (Grier, H. E et al., N.Engl. J. Med. 348, 694-701 (2003)). ESFTs contain a well characterizedchromosomal translocation. This joins the Ewing's sarcoma gene (EWS),located on chromosome 22, to an ets family gene, often friend leukemiainsertion (FLI)1 located on the chromosome 11, t(11:22) which lead tothe expression of various fusion proteins (Aykut Uren, Jeffrey ATorestsky Ewing's sarcoma oncoproteins EWS-FLI1: the perfect targetwithout a therapeutic agent, Future Oncol. 1(4), 521-528 (2005)).

In vitro and in vivo studies have demonstrated that the elimination ofthe oncoprotein, EWS-FLI1, leads to a decrease proliferation of ESTFcell lines and a decrease of tumor volume. EWS-FLI1 lacks enzymaticactivity, however, the RHA helicase A (RHA) increases EWS-FLI1-modulatedoncogenesis, therefore the protein-protein interactions between the twoproteins is required for the maintenance of the tumor growth (Hyariye NErkizan et al. A small molecule blocking oncogenic protein EWS-FlI1interacting with RHA helicase A inhibits growth of Ewing's sarcoma.Nature Medicine 15 (7) 750-756 (2009)). The paradigm of disrupting keyprotein interactions may have utility in treatment of diseases includingsarcomas with similar translocations, and leukemias with MLLtranslocations ((Heiman L J, Meltzer P. Mechanisms of sarcomadevelopment. Nat Rev Cancer 2003; 3(9):685-94); and Pui C H, Relling MV, Downing J R. Acute lymphoblastic leukemia. N Engl J Med 2004;350(15):1535-48). Moreover, disordered proteins may be excellenttherapeutic targets based on their intrinsic biochemical properties(Cheng Y, LeGall T, Oldfield C J, et al. Rational drug design viaintrinsically disordered protein. Trends Biotechnol 2006;24(10):435-42).

Despite years of in vitro and xenograft studies with antisense and siRNAdirected towards EWS-FLI1, none of these is heretofore practical as ahuman therapy based on inadequate delivery and stability. Accordingly,there is a need for improved therapies to treat disorders such as ESFTs.

SUMMARY OF THE INVENTION

In a generally applicable first aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), a compound isprovided of Formula I:

-   -   or a pharmaceutically acceptable salt thereof,    -   wherein R¹ is selected from the group consisting of hydrogen,        C₁₋₆ alkyl, one amino acid, two amino acids linked together,        three amino acids linked together,

-   -   R³, R⁴, R⁵, R⁹, R¹⁷ and R¹⁸ are each independently selected from        the group consisting of hydrogen, halogen, C₁₋₆ alkyl, C₁₋₆        alkoxy, —C(═O)NH₂, —NO₂, —NH₂, —OH, —NH(R¹⁵), —N(R¹⁵)₂, and        —SR¹⁵;    -   R¹⁰, R¹¹, R¹², and R¹³ are each independently selected from the        group consisting of hydrogen, halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,        —C(═O)NH₂, —NO₂, —NH₂, —OH, —NH(R¹⁵), —N(R¹⁵)₂, and —SR¹⁵;    -   R⁶ is C₁₋₆ dialkyl amine;    -   R⁷ is selected from the group consisting of hydrogen and C₁₋₆        alkyl;    -   R⁸ and R¹⁵ are each independently C₁₋₆ alkyl;    -   each R¹⁶ is independently hydrogen, —OH, or C₁₋₆ alkoxy;    -   n is an integer from 0 to 4;    -   p is 1 or 3; and    -   the dashed line represents an optional double bond where said        double bond has a configuration selected from the group        consisting of cis and trans,    -   with the proviso that at least one of R³, R⁴, R⁵, R⁹, and R¹⁴ is        selected from the group consisting of —NH(R¹⁵), —N(R¹⁵)₂, and        —SR¹⁵.

In an embodiment of the first aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the compound of Formula I is:

-   -   or a pharmaceutically acceptable salt thereof.

In an embodiment of the first aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the compound of Formula I is:

-   -   or a pharmaceutically acceptable salt thereof.

In an embodiment of the first aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), R¹ is selected from the group consisting of Leu,Leu-Asp, Leu-Asp-Ala, —CH₂—C(═O)—NHCH₂COOH, —CH₂—C(═O)—(CH₂)C(CH₃)₂,

In an embodiment of the first aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), R³ is selected from —NH(R¹⁵), —N(R¹⁵)₂, and —SR¹⁵.

In an embodiment of the first aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), R³ is —N(CH₃)₂.

In an embodiment of the first aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), R³ is —SCH₃.

In an embodiment of the first aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the compound of Formula I is:

-   -   or a pharmaceutically acceptable salt thereof.

In a generally applicable second aspect (i.e. independently combinablewith any of the aspects or embodiments identified herein), a method isprovided of treating a cancer comprising administering to a subject inneed thereof an effective amount of the compound of Formula I.

In an embodiment of the second aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the subject is mammalian.

In an embodiment of the second aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the subject is human.

In an embodiment of the second aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the cancer is selected from the group consisting oflung adenocarcinoma, and glioblastoma multiforme.

In an embodiment of the second aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the cancer comprises a translocation comprising anETS gene selected from the group consisting of FLI1, ETV1, ETV4, ERG,ETS1, and ETS2.

In an embodiment of the second aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the compound is administered parentally.

In a generally applicable third aspect (i.e. independently combinablewith any of the aspects or embodiments identified herein), a method isprovided of killing or inhibiting the growth of a neoplastic cellcomprising contacting the cell with an effective amount of a compound ofFormula I:

-   -   or a pharmaceutically acceptable salt thereof,    -   wherein R¹ is selected from the group consisting of hydrogen,        C₁₋₆ alkyl, one amino acid, two amino acids linked together,        three amino acids linked together,

-   -   R³, R⁴, R⁵, R⁹, and R¹⁴ are each independently selected from the        group consisting of hydrogen, halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,        —C(═O)NH₂, —NO₂, —NH₂, —OH, —NH(R¹⁵), —N(R¹⁵)₂, and —SR¹⁵;    -   R¹⁰, R¹¹, R¹², and R¹³ are each independently selected from the        group consisting of hydrogen, halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,        —C(═O)NH₂, —NO₂, —NH₂, —OH, —NH(R¹⁵), —N(R¹⁵)₂, and —SR¹⁵;    -   R⁶ is C₁₋₆ dialkyl amine;    -   R⁷ is selected from the group consisting of hydrogen and C₁₋₆        alkyl;    -   R⁸ and R¹⁵ are each independently C₁₋₆ alkyl;    -   each R¹⁶ is independently hydrogen, —OH, or C₁₋₆ alkoxy;    -   R¹⁷ and R¹⁸ are independently H or F;    -   n is an integer from 0 to 4;    -   p is 1 or 3; and    -   the dashed line represents an optional double bond where said        double bond has a configuration selected from the group        consisting of cis and trans,    -   with the proviso that at least one of R³, R⁴, R⁵, R⁹, and R¹⁴ is        selected from the group consisting of —NH(R¹⁵), —N(R¹⁵)₂, and        —SR¹⁵.

In an embodiment of the third aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the compound of Formula I is:

-   -   or a pharmaceutically acceptable salt thereof.

In an embodiment of the third aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), The compound of Formula I is:

-   -   or a pharmaceutically acceptable salt thereof.

In an embodiment of the third aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), R¹ is selected from the group consisting of Leu,Leu-Asp, Leu-Asp-Ala, —CH₂—C(═O)—NHCH₂COOH, —CH₂—C(═O)—(CH₂)C(CH₃)₂,

In an embodiment of the third aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), R³ is selected from —NH(R¹⁵), —N(R¹⁵)₂, and —SR¹⁵.

In an embodiment of the third aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), R³ is —N(CH₃)₂.

In an embodiment of the third aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), R³ is —SCH₃.

In an embodiment of the third aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), The compound of Formula I is:

-   -   or a pharmaceutically acceptable salt thereof.

In an embodiment of the third aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the cell is mammalian.

In an embodiment of the third aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the cell is human.

In an embodiment of the third aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the cell is selected from the group consisting oflung adenocarcinoma, and glioblastoma multiforme.

In an embodiment of the third aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the cell comprises a translocation comprising an ETSgene selected from the group consisting of FLI1, ETV1, ETV4, ERG, ETS1,and ETS2.

In an embodiment of the third aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the cell is in vivo.

In an embodiment of the third aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), the cell is ex vivo.

In a generally applicable fourth aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein), a compound isprovided of Formula I:

-   -   or a pharmaceutically acceptable salt thereof,    -   wherein R¹ is selected from the group consisting of hydrogen,        C₁₋₆ alkyl, one amino acid, two amino acids linked together,        three amino acids linked together,

-   -   R³, R⁴, R⁵, R⁹, and R¹⁴ are each independently selected from the        group consisting of hydrogen, halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,        —C(═O)NH₂, —NO₂, —NH₂, —OH, —NH(R¹⁵), —N(R¹⁵)₂, and —SR¹⁵;    -   R¹⁰, R¹¹, R¹², and R¹³ are each independently selected from the        group consisting of hydrogen, halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,        —C(═O)NH₂, —NO₂, —NH₂, —OH, —NH(R¹⁵), —N(R¹⁵)₂, and —SR¹⁵;    -   R⁶ is C₁₋₆ dialkyl amine;    -   R⁷ is selected from the group consisting of hydrogen and C₁₋₆        alkyl;    -   R⁸ and R¹⁵ are each independently C₁₋₆ alkyl;    -   each R¹⁶ is independently hydrogen, —OH, or C₁₋₆ alkoxy;    -   R¹⁷ and R¹⁸ are independently H or F;    -   n is an integer from 0 to 4;    -   p is 1 or 3; and    -   the dashed line represents an optional double bond where said        double bond has a configuration selected from the group        consisting of cis and trans,    -   with the proviso that at least one of R³, R⁴, R⁵, R⁹, and R¹⁴ is        selected from the group consisting of —NH(R¹⁵), —N(R¹⁵)₂, and        —SR¹⁵.

In an embodiment of the fourth aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), R′ is selected from the group consisting of Leu,Leu-Asp, Leu-Asp-Ala, —CH₂—C(═O)—NHCH₂COOH, —CH₂—C(═O)—(CH₂)C(CH₃)₂,

In an embodiment of the fourth aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), R³ is selected from —NH(R¹⁵), —N(R¹⁵)₂, and —SR¹⁵.

In an embodiment of the fourth aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), R³ is —N(CH₃)₂.

In an embodiment of the fourth aspect, which is generally applicable(i.e., independently combinable with any of the aspects or embodimentsidentified herein), R³ is —SCH₃.

In a generally applicable fifth aspect (i.e., independently combinablewith any of the aspects or embodiments identified herein),pharmaceutical compositions are provided comprising compounds of FormulaI.

Any of the features of an embodiment of the first through fifth aspectsis applicable to all aspects and embodiments identified herein.Moreover, any of the features of an embodiment of the first throughfifth aspects is independently combinable, partly or wholly with otherembodiments described herein in any way, e.g., one, two, or three ormore embodiments may be combinable in whole or in part. Further, any ofthe features of an embodiment of the first through fifth aspects may bemade optional to other aspects or embodiments. Any aspect or embodimentof a method can be performed using a compound of another aspect orembodiment, and any aspect or embodiment of a compound can be configuredto be employed in a method of another aspect or embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of NSC635437 and a generic structure forcertain analogs.

FIG. 2 shows an example strategy to increase the potency of YK-4-279.

FIG. 3A is a graph of the growth inhibition of TC71 and TC32 cells forvarious concentrations of YK-4-279 and PT-1-33. FIG. 3B is a graph ofthe growth inhibition of TC71 cells for various concentrations ofYK-4-279, PT-1-33, and PT-1-55. FIG. 3C is a graph of the growthinhibition of TC71 cells for various concentrations of YK-4-279 andPT-1-123.

FIG. 4 is a photomicrograph of an immunoblot of protein lysates fromTC32 cells treated with YK-4-279 and co-precipitated with RHA, EWS-FLI1or total protein.

FIGS. 5A-5G are graphs of the relative optical density in ELISA assaysmeasuring inhibition of EWS-FLI1 binding to RHA by various candidateagents.

FIG. 6A and FIG. 6B are graphs showing general trends for relativeluciferase activity for various concentrations of candidate agents inluciferase assays measuring inhibition of EWS-FLI1 binding to the NROB1promoter.

FIG. 7A-FIG. 7I illustrate luciferase activity for variousconcentrations of candidate agents in luciferase assays measuringinhibition of EWS-FLI1 binding to the NROB1 promoter.

FIG. 8 depicts the cBioPortal for cancer genomics website interface.

FIG. 9 depicts YK-4-279 binds to ERG and ETV1 with a KD of 11.7 μM and17.9 μM respectively with steady state kinetics measured on a BiacoreT100 instrument.

FIG. 10 depicts GBM cell lines overexpress FLI1 which correlates withYK-4-279 sensitivity. Top panel: immunoblot probed with anti-FLI1. Ewingsarcoma TC32 included as positive control. Expected size of FLI1, 50,EWS-FLI1 68 kDa. Bottom panel: graph of IC50 and densiometry.

FIG. 11 depicts GEMM overexpression of FLI1 compared with normal brain.RNA was extracted from normal and tumor tissues from control andgenetically modified mice. Hybridization followed by normalizationshowed that FLI1 probes were significantly elevated it the tumors butnot normal brain tissues.

FIG. 12 depicts GBM expression of FLI1. Immunostaining against FLI1 inhuman glioblastoma shows positive nuclear staining in many of the tumorcells as well as in vessel endothelium and inflammatory cells (40×objective).

FIGS. 13A and 13B illustrate that three days of treatment with(S)-YK-4-279 or racemic shows significant tumor regression. FIG. 13A:Mice with ES xenografts were treated with 400 mg/kg compound or controlsas indicated. Starting well-established tumors (300 mm³), mice weretreated with intraperitoneal compound for three days, 6 total doses.FIG. 13B: H and E stained tumors from same experiment.

FIG. 14 illustrates ERG induces expression of ZEB1 and ZEB2, whichactivate EMT leading to lung cancer metastasis and drug resistance.

FIG. 15 illustrates YK-4-279 directly interacts with ERG protein.Purified recombinant ERG was immobilized on Biacore CMS microchips, anddirect binding to eight different YK-4-279 concentrations (0.1-50 μM)was determined by SPR. Steady state KD was calculated usingBiaevaluation software.

FIGS. 16A and 16B illustrates that YK-4-279 inhibits transcriptionalactivity of ERG. FIG. 16A. Luciferase assays of Cos-7 cellscotransfected with ERG and an Id-2 reporter luciferase construct.YK-4-279 treatment decreased Id-2 promoter activity without affectingERG levels (*; p<0.001). FIG. 16B. VCaP cells were treated with siERG orYK-4-279 for 48 hours and ERG target mRNA and protein levels weredetermined. YK-4-279 treatment resulted in decreased PLAU, ADAM19 andPLAT mRNA expression. PLAU levels were also reduced.

FIG. 17 illustrates that NSCLC cell lines express ERG protein. Totalprotein lysates from indicated NSCLC cell lines were separated by PAGE.Expression of human ERG protein was confirmed by western blotting usingan anti-ERG antibody (upper panel). Molecular weight markers are givenon the left. Equal protein loading was confirmed by stripping andre-blotting the same membrane with an anti-beta-actin antibody (lowerpanel).

FIGS. 18A and 18B illustrate that ERG expression induces EMT markers.H358 NSCLC cells were transfected with a cDNA coding for human ERGprotein. Increased ERG expression was detected by western blotting (FIG.18A). Real-time PCR analysis revealed higher expression of ZEB1 andFOXC2 in ERG expressing cells (FIG. 18B). Data is first normalized for18S RNA and then expressed as fold induction over empty vectortransfected cells.

FIG. 19 illustrates that YK-4-279 inhibits EMT in NSCLC. A549 cellsexpressed higher levels of ZEB1 and FOXC2 with increased TGF-βexpression and reduced levels with ERG inhibition by YK-4-279. Data wasfirst normalized to 18S RNA and then fold expression calculated bydividing to control for each group.

DETAILED DESCRIPTION

The following description and examples illustrate some exemplaryembodiments of the disclosed invention in detail. Those of skill in theart will recognize that there are numerous variations and modificationsof this invention that are encompassed by its scope. Accordingly, thedescription of a certain exemplary embodiment should not be deemed tolimit the scope of the present invention.

A NCI/DTP library of three thousands small molecules was screened forEWS-FLI1 binding using Surface Plasmon Resonance. The compound,NSC635437, was selected as a suitable candidate for further optimizationand further study (FIG. 1). Of the first series of analogs designed,YK-4-279, was the most active (FIG. 2). YK-4-279 has been shown tofunctionally inhibit EWS-FLI1 and ESFT cells and leads to caspase-3activity increase (Hyariye N Erkizan et al. A small molecule blockingoncogenic protein EWS-FlI1 interacting with RHA helicase A inhibitsgrowth of Ewing's sarcoma. Nature Medicine 15(7) 750-756 (2009)). Thepresent application relates to improved compounds and methods of usingsuch compounds to treat disorders such as lung adenocarcinoma,glioblastoma multiforme, and cancers comprising a translocationcomprising an ETS gene selected from the group consisting of FLI1, ETV1,ETV4, ERG, ETS1, and ETS2.

Other methods and compositions useful with those provided herein aredisclosed in Int. Pub. No. WO 2008/083326; U.S. Pub. No. 2010/0167994;U.S. Prov App. No. 61/623,349; and Int. Pub. No. WO 2013/155341, thedisclosures of which are expressly incorporated herein by reference intheir entireties.

DEFINITIONS

As used herein, any “R” group(s) such as, without limitation, R, R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R^(a), R^(b),represent substituents that can be attached to the indicated atom. An Rgroup may be substituted or unsubstituted. If two “R” groups aredescribed as being “taken together” the R groups and the atoms they areattached to can form a cycloalkyl, aryl, heteroaryl, or heterocycle. Forexample, without limitation, if R^(1a) and R^(1b) of an NR^(1a)R^(1b)group are indicated to be “taken together,” it means that they arecovalently bonded to one another to form a ring:

Whenever a group is described as being “optionally substituted” thatgroup may be unsubstituted or substituted with one or more of theindicated substituents. Likewise, when a group is described as being“unsubstituted or substituted” if substituted, the substituent(s) may beselected from one or more the indicated substituents. If no substituentsare indicated, it is meant that the indicated “optionally substituted”or “substituted” group may be substituted with one or more group(s)individually and independently selected from alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl,hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio,arylthio, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido,N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato,thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl,haloalkyl, haloalkoxy, trihalomethanesulfonyl,trihalomethanesulfonamido, an amino, a mono-substituted amino and adi-substituted amino group, and protected derivatives thereof.

As used herein, “C_(a) to C_(b)” in which “a” and “b” are integers referto the number of carbon atoms in an alkyl, alkenyl or alkynyl group, orthe number of carbon atoms in the ring of a cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl or heteroalicyclyl group. That is, thealkyl, alkenyl, alkynyl, ring of the cycloalkyl, ring of thecycloalkenyl, ring of the cycloalkynyl, ring of the aryl, ring of theheteroaryl or ring of the heteroalicyclyl can contain from “a” to “b”,inclusive, carbon atoms. Thus, for example, a “C₁ to C₄ alkyl” grouprefers to all alkyl groups having from 1 to 4 carbons, that is, CH₃—,CH₃CH₂—, CH₃CH₂CH₂—, (CH₃)₂CH—, CH₃CH₂CH₂CH₂—, CH₃CH₂CH(CH₃)— and(CH₃)₃C—. If no “a” and “b” are designated with regard to an alkyl,alkenyl, alkynyl, cycloalkyl cycloalkenyl, cycloalkynyl, aryl,heteroaryl or heteroalicyclyl group, the broadest range described inthese definitions is to be assumed.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that includes a fully saturated (no double or triple bonds)hydrocarbon group. The alkyl group may have 1 to 20 carbon atoms(whenever it appears herein, a numerical range such as “1 to 20” refersto each integer in the given range; e.g., “1 to 20 carbon atoms” meansthat the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3carbon atoms, etc., up to and including 20 carbon atoms, although thepresent definition also covers the occurrence of the term “alkyl” whereno numerical range is designated). The alkyl group may also be a mediumsize alkyl having 1 to 10 carbon atoms. The alkyl group could also be alower alkyl having 1 to 6 carbon atoms. The alkyl group of the compoundsmay be designated as “C₁-C₄ alkyl” or similar designations. By way ofexample only, “C₁-C₄ alkyl” indicates that there are one to four carbonatoms in the alkyl chain, i.e., the alkyl chain is selected from methyl,ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl.Typical alkyl groups include, but are in no way limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl andhexyl. The alkyl group may be substituted or unsubstituted.

As used herein, “alkenyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more double bonds. Analkenyl group may be unsubstituted or substituted.

As used herein, “alkynyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more triple bonds. Analkynyl group may be unsubstituted or substituted.

As used herein, “cycloalkyl” refers to a completely saturated (no doubleor triple bonds) mono- or multi-cyclic hydrocarbon ring system. Whencomposed of two or more rings, the rings may be joined together in afused fashion. Cycloalkyl groups can contain 3 to 10 atoms in thering(s) or 3 to 8 atoms in the ring(s). A cycloalkyl group may beunsubstituted or substituted. Typical cycloalkyl groups include, but arein no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and cyclooctyl.

As used herein, “cycloalkenyl” refers to a mono- or multi-cyclichydrocarbon ring system that contains one or more double bonds in atleast one ring; although, if there is more than one, the double bondscannot form a fully delocalized pi-electron system throughout all therings (otherwise the group would be “aryl,” as defined herein). Whencomposed of two or more rings, the rings may be connected together in afused fashion. A cycloalkenyl group may be unsubstituted or substituted.

As used herein, “cycloalkynyl” refers to a mono- or multi-cyclichydrocarbon ring system that contains one or more triple bonds in atleast one ring. If there is more than one triple bond, the triple bondscannot form a fully delocalized pi-electron system throughout all therings. When composed of two or more rings, the rings may be joinedtogether in a fused fashion. A cycloalkynyl group may be unsubstitutedor substituted.

As used herein, “aryl” refers to a carbocyclic (all carbon) monocyclicor multicyclic aromatic ring system (including fused ring systems wheretwo carbocyclic rings share a chemical bond) that has a fullydelocalized pi-electron system throughout all the rings. The number ofcarbon atoms in an aryl group can vary. For example, the aryl group canbe a C₆-C₁₄ aryl group, a C₆-C₁₀ aryl group, or a C₆ aryl group.Examples of aryl groups include, but are not limited to, benzene,naphthalene and azulene. An aryl group may be substituted orunsubstituted.

As used herein, “heteroaryl” refers to a monocyclic or multicyclicaromatic ring system (a ring system with fully delocalized pi-electronsystem) that contain(s) one or more heteroatoms, that is, an elementother than carbon, including but not limited to, nitrogen, oxygen andsulfur. The number of atoms in the ring(s) of a heteroaryl group canvary. For example, the heteroaryl group can contain 4 to 14 atoms in thering(s), 5 to 10 atoms in the ring(s) or 5 to 6 atoms in the ring(s).Furthermore, the term “heteroaryl” includes fused ring systems where tworings, such as at least one aryl ring and at least one heteroaryl ring,or at least two heteroaryl rings, share at least one chemical bond.Examples of heteroaryl rings include, but are not limited to, furan,furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole,benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole,1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole,benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole,benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole,tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine,pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline,and triazine. A heteroaryl group may be substituted or unsubstituted.

As used herein, “heterocyclyl” or “heteroalicyclyl” refers to three-,four-, five-, six-, seven-, eight-, nine-, ten-, up to 18-memberedmonocyclic, bicyclic, and tricyclic ring system wherein carbon atomstogether with from 1 to 5 heteroatoms constitute said ring system. Aheterocycle may optionally contain one or more unsaturated bondssituated in such a way, however, that a fully delocalized pi-electronsystem does not occur throughout all the rings. The heteroatom(s) is anelement other than carbon including, but not limited to, oxygen, sulfur,and nitrogen. A heterocycle may further contain one or more carbonyl orthiocarbonyl functionalities, so as to make the definition includeoxo-systems and thio-systems such as lactams, lactones, cyclic imides,cyclic thioimides and cyclic carbamates. When composed of two or morerings, the rings may be joined together in a fused fashion.Additionally, any nitrogens in a heteroalicyclic may be quaternized.Heterocyclyl or heteroalicyclic groups may be unsubstituted orsubstituted. Examples of such “heterocyclyl” or “heteroalicyclyl” groupsinclude but are not limited to, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane,1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane,1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiole, 1,3-dithiolane,1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide,succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine,hydantoin, dihydrouracil, trioxane, hexahydro-1,3,5-triazine,imidazoline, imidazolidine, isoxazoline, isoxazolidine, oxazoline,oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholine,oxirane, piperidine N-Oxide, piperidine, piperazine, pyrrolidine,pyrrolidone, pyrrolidione, 4-piperidone, pyrazoline, pyrazolidine,2-oxopyrrolidine, tetrahydropyran, 4H-pyran, tetrahydrothiopyran,thiamorpholine, thiamorpholine sulfoxide, thiamorpholine sulfone, andtheir benzo-fused analogs (e.g., benzimidazolidinone,tetrahydroquinoline, 3,4-methylenedioxyphenyl).

As used herein, “aralkyl” and “aryl(alkyl)” refer to an aryl groupconnected, as a substituent, via a lower alkylene group. The loweralkylene and aryl group of an aralkyl may be substituted orunsubstituted. Examples include but are not limited to benzyl,2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

As used herein, “heteroaralkyl” and “heteroaryl(alkyl)” refer to aheteroaryl group connected, as a substituent, via a lower alkylenegroup. The lower alkylene and heteroaryl group of heteroaralkyl may besubstituted or unsubstituted. Examples include but are not limited to2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl,pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl, and theirbenzo-fused analogs.

A “(heteroalicyclyl)alkyl” and “(heterocyclyl)alkyl” refer to aheterocyclic or a heteroalicyclylic group connected, as a substituent,via a lower alkylene group. The lower alkylene and heterocyclyl of a(heteroalicyclyl)alkyl may be substituted or unsubstituted. Examplesinclude but are not limited tetrahydro-2H-pyran-4-yl)methyl,(piperidin-4-yl)ethyl, (piperidin-4-yl)propyl,(tetrahydro-2H-thiopyran-4-yl)methyl, and (1,3-thiazinan-4-yl)methyl.

“Lower alkylene groups” are straight-chained —CH₂— tethering groups,forming bonds to connect molecular fragments via their terminal carbonatoms. Examples include but are not limited to methylene (—CH₂—),ethylene (—CH₂CH₂—), propylene (—CH₂CH₂CH₂—), and butylene(—CH₂CH₂CH₂CH₂—). A lower alkylene group can be substituted by replacingone or more hydrogen of the lower alkylene group with a substituent(s)listed under the definition of “substituted.”

As used herein, “alkoxy” refers to the formula —OR wherein R is analkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl or acycloalkynyl is defined as above. A non-limiting list of alkoxys ismethoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy,iso-butoxy, sec-butoxy and tert-butoxy. An alkoxy may be substituted orunsubstituted.

As used herein, “acyl” refers to a hydrogen, alkyl, alkenyl, alkynyl, oraryl connected, as substituents, via a carbonyl group. Examples includeformyl, acetyl, propanoyl, benzoyl, and acryl. An acyl may besubstituted or unsubstituted.

As used herein, “hydroxyalkyl” refers to an alkyl group in which one ormore of the hydrogen atoms are replaced by a hydroxy group. Exemplaryhydroxyalkyl groups include but are not limited to, 2-hydroxyethyl,3-hydroxypropyl, 2-hydroxypropyl, and 2,2-dihydroxyethyl. A hydroxyalkylmay be substituted or unsubstituted.

As used herein, “haloalkyl” refers to an alkyl group in which one ormore of the hydrogen atoms are replaced by a halogen (e.g.,mono-haloalkyl, di-haloalkyl and tri-haloalkyl). Such groups include butare not limited to, chloromethyl, fluoromethyl, difluoromethyl,trifluoromethyl and 1-chloro-2-fluoromethyl, 2-fluoroisobutyl. Ahaloalkyl may be substituted or unsubstituted.

As used herein, “haloalkoxy” refers to an alkoxy group in which one ormore of the hydrogen atoms are replaced by a halogen (e.g.,mono-haloalkoxy, di-haloalkoxy and tri-haloalkoxy). Such groups includebut are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy,trifluoromethoxy, 1-chloro-2-fluoromethoxy, and 2-fluoroisobutoxy. Ahaloalkoxy may be substituted or unsubstituted.

As used herein, “aryloxy” and “arylthio” refers to RO— and RS—, in whichR is an aryl, such as but not limited to phenyl. Both an aryloxy andarylthio may be substituted or unsubstituted.

A “sulfenyl” or “thio” group refers to an “—SR” group in which R can behydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or(heteroalicyclyl)alkyl. A sulfenyl may be substituted or unsubstituted.The term “sulfenyl” or “thio” includes, but is not limited to an —SHgroup (also referred to as a “thiol” group) as well as an —SR_(A) group(also referred to as a “thioether” when R_(A) is not hydrogen).

A “sulfinyl” group refers to an “—S(═O)—R” group in which R can be thesame as defined with respect to sulfenyl. A sulfinyl may be substitutedor unsubstituted.

A “sulfonyl” group refers to an “SO₂R” group in which R can be the sameas defined with respect to sulfenyl. A sulfonyl may be substituted orunsubstituted.

An “O-carboxy” group refers to a “RC(═O)O—” group in which R can behydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, or(heteroalicyclyl)alkyl, as defined herein. An O-carboxy may besubstituted or unsubstituted.

The terms “ester” and “C-carboxy” refer to a “—C(═O)OR” group in which Rcan be the same as defined with respect to O-carboxy. An ester andC-carboxy may be substituted or unsubstituted.

A “thiocarbonyl” group refers to a “—C(═S)R” group in which R can be thesame as defined with respect to O-carboxy. A thiocarbonyl may besubstituted or unsubstituted.

A “trihalomethanesulfonyl” group refers to an “X₃CSO₂—” group wherein Xis a halogen.

A “trihalomethanesulfonamido” group refers to an “X₃CS(O)₂N(R_(A))”group wherein X is a halogen and R_(A) is hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl.

The term “amino” as used herein refers to a —N(R)₂ group, wherein R isindependently selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. Anamino may be substituted or unsubstituted. The term “amino” includes,but is not limited to a —NH₂ group (also referred to as an “ammonium”group), a —NHR group (also referred to as a “secondary amine” when R isnot hydrogen), or a —NR₂ group (also referred to as a “tertiary amine”when R is not hydrogen).

As used herein, the term “hydroxy” refers to a —OH group.

A “cyano” group refers to a “—CN” group.

The term “azido” as used herein refers to a —N₃ group.

An “isocyanato” group refers to a “—NCO” group.

A “thiocyanato” group refers to a “—CNS” group.

An “isothiocyanato” group refers to an “—NCS” group.

A “mercapto” group refers to an “—SH” group.

A “carbonyl” group refers to a C═O group.

An “S-sulfonamido” group refers to a “—SO₂N(R_(A)R_(B))” group in whichR_(A) and R_(B) can be independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An S-sulfonamidomay be substituted or unsubstituted.

An “N-sulfonamido” group refers to a “RSO₂N(R_(A))—” group in which Rand R_(A) can be independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An N-sulfonamidomay be substituted or unsubstituted.

An “O-carbamyl” group refers to a “—OC(═O)N(R_(A)R_(B))” group in whichR_(A) and R_(B) can be independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An O-carbamyl maybe substituted or unsubstituted.

An “N-carbamyl” group refers to an “ROC(═O)N(R_(A))—” group in which Rand R_(A) can be independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An N-carbamyl maybe substituted or unsubstituted.

An “O-thiocarbamyl” group refers to a “—OC(═S)—N(R_(A)R_(B))” group inwhich R_(A) and R_(B) can be independently hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An O-thiocarbamylmay be substituted or unsubstituted.

An “N-thiocarbamyl” group refers to an “ROC(═S)N(R_(A))—” group in whichR and R_(A) can be independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An N-thiocarbamylmay be substituted or unsubstituted.

A “C-amido” group refers to a “—C(═O)N(R_(A)R_(B))” group in which R_(A)and R_(B) can be independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. A C-amido may besubstituted or unsubstituted.

An “N-amido” group refers to a “RC(═O)N(R_(A))—” group in which R andR_(A) can be independently hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An N-amido may besubstituted or unsubstituted.

The term “halogen atom” or “halogen” as used herein, means any one ofthe radio-stable atoms of column 7 of the Periodic Table of theElements, such as, fluorine, chlorine, bromine and iodine.

Where the numbers of substituents is not specified (e.g. haloalkyl),there may be one or more substituents present. For example “haloalkyl”may include one or more of the same or different halogens. As anotherexample, “C₁-C₃ alkoxyphenyl” may include one or more of the same ordifferent alkoxy groups containing one, two or three atoms.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (See, Biochem. 11:942-944(1972)).

It is understood that the compounds described herein can be labeledisotopically. Substitution with isotopes such as deuterium may affordcertain therapeutic advantages resulting from greater metabolicstability, such as, for example, increased in vivo half-life or reduceddosage requirements. Each chemical element as represented in a compoundstructure may include any isotope of said element. For example, in acompound structure a hydrogen atom may be explicitly disclosed orunderstood to be present in the compound. At any position of thecompound that a hydrogen atom may be present, the hydrogen atom can beany isotope of hydrogen, including but not limited to hydrogen-1(protium) and hydrogen-2 (deuterium). Thus, reference herein to acompound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

It is understood that the methods and combinations described hereininclude crystalline forms (also known as polymorphs, which include thedifferent crystal packing arrangements of the same elemental compositionof a compound), amorphous phases, salts, solvates, and hydrates. In someembodiments, the compounds described herein exist in solvated forms withpharmaceutically acceptable solvents such as water, ethanol, or thelike. In other embodiments, the compounds described herein exist inunsolvated form. Solvates contain either stoichiometric ornon-stoichiometric amounts of a solvent, and may be formed during theprocess of crystallization with pharmaceutically acceptable solventssuch as water, ethanol, or the like. Hydrates are formed when thesolvent is water, or alcoholates are formed when the solvent is alcohol.In addition, the compounds provided herein can exist in unsolvated aswell as solvated forms. In general, the solvated forms are consideredequivalent to the unsolvated forms for the purposes of the compounds andmethods provided herein.

Where a range of values is provided, it is understood that the upper andlower limit, and each intervening value between the upper and lowerlimit of the range is encompassed within the embodiments.

Certain Synthetic Methods

In some embodiments, appropriate acetophenone (4.0 equiv.) and catalyticamount of diethylamine (10 drops) were added to a solution of4,7-dichloroisatin (1.0 equiv.) in methanol (5 mL). The mixture wasstirred at room temperature until starting material (4,7-dichloroisatin)disappeared completely. The resulted solution was concentrated andapplied to flash chromatography eluting with Hexane/Ethyl acetate toafford pure product in quantitative yield. Further purification was doneby recrystallization with Hexane/Ethyl acetate. NMR spectra wererecorded using a Varian-400 spectrometer for ¹H (400 MHz), chemicalshifts (δ) are given in ppm downfield from tetramethylsilane as internalstandard, and coupling constants (J-values) are in hertz (Hz). Elementalanalyses were performed by Atlantic Microlabs.

Certain compounds provided herein can be prepared according to thefollowing synthesis schemes.

In these schemes, ketone (4.0 equiv.) and a catalytic amount ofdiethylamine (10 drops) are added to a solution of substituted isatin(1.0 equiv.) in methanol (5 mL). The mixture is stirred at roomtemperature until starting material (substituted isatin) disappearscompletely. The resulting solution is concentrated and applied to flashchromatography eluting with hexane/ethyl acetate to afford pure productin quantitative yield. Further purification is done by recrystallizationwith hexane/ethyl acetate.

The inhibitors incorporating a carbon-carbon double bond in the grouplinking the two ring systems can be prepared from the correspondingsaturated inhibitor by reducing the compound using synthetic techniquesknown in the art.

Certain Compounds

Certain compounds provided herein include compounds having a Formula I:

or a pharmaceutically acceptable salt thereof, wherein R¹ is selectedfrom the group consisting of hydrogen, C₁₋₆ alkyl, one amino acid, twoamino acids linked together, three amino acids linked together,

R³, R⁴, R⁵, R⁹, R¹⁴, R¹⁷ and R¹⁸ are each independently selected fromthe group consisting of hydrogen, halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy,—C(═O)NH₂, —NO₂, —NH₂, —OH—NH(R¹⁵), —N(R¹⁵)₂, and —SR¹⁵; R¹⁰, R¹², andR¹³ are each independently selected from the group consisting ofhydrogen, halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, —C(═O)NH₂, —NO₂, —NH₂, —OH,—NH(R¹⁵), —N(R¹⁵)₂, and —SR¹⁵; R⁶ is C₁₋₆ dialkyl amine; R⁷ is selectedfrom the group consisting of hydrogen and C₁₋₆ alkyl; R⁸ and R¹⁵ areeach independently C₁₋₆ alkyl; each R¹⁶ is independently hydrogen, —OH,or C₁₋₆ alkoxy; n is an integer from 0 to 4; p is 1 or 3; and the dashedline represents an optional double bond where said double bond has aconfiguration selected from the group consisting of cis and trans, withthe proviso that at least one of R³, R⁴, R⁵, R⁹, and R¹⁴ is selectedfrom the group consisting of —NH(R¹⁵), —N(R¹⁵)₂, and —SR¹⁵.

In some embodiments, The compound of Formula I is:

-   -   or a pharmaceutically acceptable salt thereof.

In some embodiments, The compound of Formula I is:

-   -   or a pharmaceutically acceptable salt thereof.

In some embodiments, R′ is selected from the group consisting of Leu,Leu-Asp, Leu-Asp-Ala, —CH₂—C(═O)—NHCH₂COOH, —CH₂—C(═O)—(CH₂)C(CH₃)₂,

In some embodiments, R³ is selected from —NH(R¹⁵), —N(R¹⁵)₂, and —SR¹⁵.

In some embodiments, R³ is —N(CH₃)₂.

In some embodiments, R³ is —SCH₃.

In some embodiments, The compound of Formula I is:

-   -   or a pharmaceutically acceptable salt thereof.

Depending upon the substituents present, the small molecule inhibitorscan be in a form of a pharmaceutically acceptable salt. The terms“pharmaceutically acceptable salt” as used herein are broad terms, andis to be given its ordinary and customary meaning to a person ofordinary skill in the art (and is not to be limited to a special orcustomized meaning), and refers without limitation to salts preparedfrom pharmaceutically acceptable, non-toxic acids or bases. Suitablepharmaceutically acceptable salts include metallic salts, e.g., salts ofaluminum, zinc, alkali metal salts such as lithium, sodium, andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts; organic salts, e.g., salts of lysine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine), procaine, and tris;salts of free acids and bases; inorganic salts, e.g., sulfate,hydrochloride, and hydrobromide; and other salts which are currently inwidespread pharmaceutical use and are listed in sources well known tothose of skill in the art, such as, for example, The Merck Index. Anysuitable constituent can be selected to make a salt of the therapeuticagents discussed herein, provided that it is non-toxic and does notsubstantially interfere with the desired activity.

The compounds of preferred embodiments can include isomers, racemates,optical isomers, enantiomers, diastereomers, tautomers, and cis/transconformers. All such isomeric forms are included within preferredembodiments, including mixtures thereof. As discussed above, thecompounds of preferred embodiments may have chiral centers, for example,they may contain asymmetric carbon atoms and may thus exist in the formof enantiomers or diastereoisomers and mixtures thereof, e.g.,racemates. Asymmetric carbon atom(s) can be present in the (R)-, (S)-,or (R,S)-configuration, preferably in the (R)- or (S)-configuration, orcan be present as mixtures. Isomeric mixtures can be separated, asdesired, according to conventional methods to obtain pure isomers.

The compounds can be in amorphous form, or in crystalline forms. Thecrystalline forms of the compounds of preferred embodiments can exist aspolymorphs, which are included in preferred embodiments. In addition,some of the compounds of preferred embodiments may also form solvateswith water or other organic solvents. Such solvates are similarlyincluded within the scope of the preferred embodiments.

Certain Pharmaceutical Compositions

It is generally preferred to administer the inhibitors of preferredembodiments in an intravenous or subcutaneous unit dosage form; however,other routes of administration are also contemplated. Contemplatedroutes of administration include but are not limited to oral,parenteral, intravenous, and subcutaneous. The inhibitors of preferredembodiments can be formulated into liquid preparations for, e.g., oraladministration. Suitable forms include suspensions, syrups, elixirs, andthe like. Particularly preferred unit dosage forms for oraladministration include tablets and capsules. Unit dosage formsconfigured for administration once a day are particularly preferred;however, in certain embodiments it can be desirable to configure theunit dosage form for administration twice a day, or more.

The pharmaceutical compositions of preferred embodiments are preferablyisotonic with the blood or other body fluid of the recipient. Theisotonicity of the compositions can be attained using sodium tartrate,propylene glycol or other inorganic or organic solutes. Sodium chlorideis particularly preferred. Buffering agents can be employed, such asacetic acid and salts, citric acid and salts, boric acid and salts, andphosphoric acid and salts. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like.

Viscosity of the pharmaceutical compositions can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the thickening agentselected. An amount is preferably used that will achieve the selectedviscosity. Viscous compositions are normally prepared from solutions bythe addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf life of the pharmaceutical compositions. Benzyl alcohol can besuitable, although a variety of preservatives including, for example,parabens, thimerosal, chlorobutanol, or benzalkonium chloride can alsobe employed. A suitable concentration of the preservative is typicallyfrom about 0.02% to about 2% based on the total weight of thecomposition, although larger or smaller amounts can be desirabledepending upon the agent selected. Reducing agents, as described above,can be advantageously used to maintain good shelf life of theformulation.

The inhibitors of preferred embodiments can be in admixture with asuitable carrier, diluent, or excipient such as sterile water,physiological saline, glucose, or the like, and can contain auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,gelling or viscosity enhancing additives, preservatives, flavoringagents, colors, and the like, depending upon the route of administrationand the preparation desired. See, e.g., “Remington: The Science andPractice of Pharmacy”, Lippincott Williams & Wilkins; 20th edition (Jun.1, 2003) and “Remington's Pharmaceutical Sciences,” Mack Pub. Co.;18^(th) and 19^(th) editions (December 1985, and June 1990,respectively). Such preparations can include complexing agents, metalions, polymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels, dextran, and the like, liposomes, microemulsions, micelles,unilamellar or multilamellar vesicles, erythrocyte ghosts orspheroblasts. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. The presence of suchadditional components can influence the physical state, solubility,stability, rate of in vivo release, and rate of in vivo clearance, andare thus chosen according to the intended application, such that thecharacteristics of the carrier are tailored to the selected route ofadministration.

For oral administration, the pharmaceutical compositions can be providedas a tablet, aqueous or oil suspension, dispersible powder or granule,emulsion, hard or soft capsule, syrup or elixir. Compositions intendedfor oral use can be prepared according to any method known in the artfor the manufacture of pharmaceutical compositions and can include oneor more of the following agents: sweeteners, flavoring agents, coloringagents and preservatives. Aqueous suspensions can contain the activeingredient in admixture with excipients suitable for the manufacture ofaqueous suspensions.

Formulations for oral use can also be provided as hard gelatin capsules,wherein the active ingredient(s) are mixed with an inert solid diluent,such as calcium carbonate, calcium phosphate, or kaolin, or as softgelatin capsules. In soft capsules, the inhibitors can be dissolved orsuspended in suitable liquids, such as water or an oil medium, such aspeanut oil, olive oil, fatty oils, liquid paraffin, or liquidpolyethylene glycols. Stabilizers and microspheres formulated for oraladministration can also be used. Capsules can include push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredient in admixture with fillers such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers.

Tablets can be uncoated or coated by known methods to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period of time. For example, atime delay material such as glyceryl monostearate can be used. Whenadministered in solid form, such as tablet form, the solid formtypically comprises from about 0.001 wt. % or less to about 50 wt. % ormore of active ingredient(s), preferably from about 0.005, 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, or 1 wt. % to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30, 35, 40, or 45 wt. %.

Tablets can contain the active ingredients in admixture with non-toxicpharmaceutically acceptable excipients including inert materials. Forexample, a tablet can be prepared by compression or molding, optionally,with one or more additional ingredients. Compressed tablets can beprepared by compressing in a suitable machine the active ingredients ina free-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding, in a suitable machine, a mixtureof the powdered inhibitor moistened with an inert liquid diluent.

Preferably, each tablet or capsule contains from about 1 mg or less toabout 1,000 mg or more of an inhibitor of the preferred embodiments,more preferably from about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mgto about 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, or 900 mg. Most preferably, tablets or capsules are providedin a range of dosages to permit divided dosages to be administered. Adosage appropriate to the patient and the number of doses to beadministered daily can thus be conveniently selected. In certainembodiments it can be preferred to incorporate two or more of thetherapeutic agents to be administered into a single tablet or otherdosage form (e.g., in a combination therapy); however, in otherembodiments it can be preferred to provide the therapeutic agents inseparate dosage forms.

Suitable inert materials include diluents, such as carbohydrates,mannitol, lactose, anhydrous lactose, cellulose, sucrose, modifieddextrans, starch, and the like, or inorganic salts such as calciumtriphosphate, calcium phosphate, sodium phosphate, calcium carbonate,sodium carbonate, magnesium carbonate, and sodium chloride.Disintegrants or granulating agents can be included in the formulation,for example, starches such as corn starch, alginic acid, sodium starchglycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin,sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose,natural sponge and bentonite, insoluble cationic exchange resins,powdered gums such as agar, karaya or tragacanth, or alginic acid orsalts thereof.

Binders can be used to form a hard tablet. Binders include materialsfrom natural products such as acacia, tragacanth, starch and gelatin,methyl cellulose, ethyl cellulose, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, and the like.

Lubricants, such as stearic acid or magnesium or calcium salts thereof,polytetrafluoroethylene, liquid paraffin, vegetable oils and waxes,sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol,starch, talc, pyrogenic silica, hydrated silicoaluminate, and the like,can be included in tablet formulations.

Surfactants can also be employed, for example, anionic detergents suchas sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctylsodium sulfonate, cationic such as benzalkonium chloride or benzethoniumchloride, or nonionic detergents such as polyoxyethylene hydrogenatedcastor oil, glycerol monostearate, polysorbates, sucrose fatty acidester, methyl cellulose, or carboxymethyl cellulose.

Controlled release formulations can be employed wherein the amifostineor analog(s) thereof is incorporated into an inert matrix that permitsrelease by either diffusion or leaching mechanisms. Slowly degeneratingmatrices can also be incorporated into the formulation. Other deliverysystems can include timed release, delayed release, or sustained releasedelivery systems.

Coatings can be used, for example, nonenteric materials such as methylcellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethylcellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose,sodium carboxy-methyl cellulose, providone and the polyethylene glycols,or enteric materials such as phthalic acid esters. Dyestuffs or pigmentscan be added for identification or to characterize differentcombinations of inhibitor doses

When administered orally in liquid form, a liquid carrier such as water,petroleum, oils of animal or plant origin such as peanut oil, mineraloil, soybean oil, or sesame oil, or synthetic oils can be added to theactive ingredient(s). Physiological saline solution, dextrose, or othersaccharide solution, or glycols such as ethylene glycol, propyleneglycol, or polyethylene glycol are also suitable liquid carriers. Thepharmaceutical compositions can also be in the form of oil-in-wateremulsions. The oily phase can be a vegetable oil, such as olive orarachis oil, a mineral oil such as liquid paraffin, or a mixturethereof. Suitable emulsifying agents include naturally-occurring gumssuch as gum acacia and gum tragacanth, naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids and hexitol anhydrides, such as sorbitan mono-oleate, andcondensation products of these partial esters with ethylene oxide, suchas polyoxyethylene sorbitan mono-oleate. The emulsions can also containsweetening and flavoring agents.

Pulmonary delivery can also be employed. The compound is delivered tothe lungs while inhaling and traverses across the lung epithelial liningto the blood stream. A wide range of mechanical devices designed forpulmonary delivery of therapeutic products can be employed, includingbut not limited to nebulizers, metered dose inhalers, and powderinhalers, all of which are familiar to those skilled in the art. Thesedevices employ formulations suitable for the dispensing of compound.Typically, each formulation is specific to the type of device employedand can involve the use of an appropriate propellant material, inaddition to diluents, adjuvants, and/or carriers useful in therapy.

The compound and/or other optional active ingredients are advantageouslyprepared for pulmonary delivery in particulate form with an averageparticle size of from 0.1 μm or less to 10 μm or more, more preferablyfrom about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 μm to about 1.0,1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0,8.5, 9.0, or 9.5 μm. Pharmaceutically acceptable carriers for pulmonarydelivery of inhibitor include carbohydrates such as trehalose, mannitol,xylitol, sucrose, lactose, and sorbitol. Other ingredients for use informulations can include DPPC, DOPE, DSPC, and DOPC. Natural orsynthetic surfactants can be used, including polyethylene glycol anddextrans, such as cyclodextran. Bile salts and other related enhancers,as well as cellulose and cellulose derivatives, and amino acids can alsobe used. Liposomes, microcapsules, microspheres, inclusion complexes,and other types of carriers can also be employed.

Pharmaceutical formulations suitable for use with a nebulizer, eitherjet or ultrasonic, typically comprise the inhibitor dissolved orsuspended in water at a concentration of about 0.01 or less to 100 mg ormore of inhibitor per mL of solution, preferably from about 0.1, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 mg to about 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, or 90 mg per mL of solution. The formulationcan also include a buffer and a simple sugar (e.g., for proteinstabilization and regulation of osmotic pressure). The nebulizerformulation can also contain a surfactant, to reduce or prevent surfaceinduced aggregation of the inhibitor caused by atomization of thesolution in forming the aerosol.

Formulations for use with a metered-dose inhaler device generallycomprise a finely divided powder containing the active ingredientssuspended in a propellant with the aid of a surfactant. The propellantcan include conventional propellants, such as chlorofluorocarbons,hydrochlorofluorocarbons, hydrofluorocarbons, and hydrocarbons.Preferred propellants include trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol,1,1,1,2-tetrafluoroethane, and combinations thereof. Suitablesurfactants include sorbitan trioleate, soya lecithin, and oleic acid.

Formulations for dispensing from a powder inhaler device typicallycomprise a finely divided dry powder containing inhibitor, optionallyincluding a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol in an amount that facilitates dispersal of thepowder from the device, typically from about 1 wt. % or less to 99 wt. %or more of the formulation, preferably from about 5, 10, 15, 20, 25, 30,35, 40, 45, or 50 wt. % to about 55, 60, 65, 70, 75, 80, 85, or 90 wt. %of the formulation.

When a compound of the preferred embodiments is administered byintravenous, parenteral, or other injection, it is preferably in theform of a pyrogen-free, parenterally acceptable aqueous solution oroleaginous suspension. Suspensions can be formulated according tomethods well known in the art using suitable dispersing or wettingagents and suspending agents. The preparation of acceptable aqueoussolutions with suitable pH, isotonicity, stability, and the like, iswithin the skill in the art. A preferred pharmaceutical composition forinjection preferably contains an isotonic vehicle such as1,3-butanediol, water, isotonic sodium chloride solution, Ringer'ssolution, dextrose solution, dextrose and sodium chloride solution,lactated Ringer's solution, or other vehicles as are known in the art.In addition, sterile fixed oils can be employed conventionally as asolvent or suspending medium. For this purpose, any bland fixed oil canbe employed including synthetic mono or diglycerides. In addition, fattyacids such as oleic acid can likewise be used in the formation ofinjectable preparations. The pharmaceutical compositions can alsocontain stabilizers, preservatives, buffers, antioxidants, or otheradditives known to those of skill in the art.

The duration of the injection can be adjusted depending upon variousfactors, and can comprise a single injection administered over thecourse of a few seconds or less, to 0.5, 0.1, 0.25, 0.5, 0.75, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 hours or more of continuous intravenous administration.

The compounds of the preferred embodiments can additionally employadjunct components conventionally found in pharmaceutical compositionsin their art-established fashion and at their art-established levels.Thus, for example, the compositions can contain additional compatiblepharmaceutically active materials for combination therapy (such assupplementary antimicrobials, antipruritics, astringents, localanesthetics, anti-inflammatory agents, reducing agents,chemotherapeutics and the like), or can contain materials useful inphysically formulating various dosage forms of the preferredembodiments, such as excipients, dyes, thickening agents, stabilizers,preservatives or antioxidants. Anti-cancer agents that can be used incombination with the compounds of preferred embodiments include, but arenot limited to, vinca alkaloids such as vinblastine and vincristine;anthracyclines such as doxorubicin, daunorubicin, epirubicin;anthracenes such as bisantrene and mitoxantrone; epipodophyllo-toxinssuch as etoposide and teniposide; and other anticancer drugs such asactinomyocin D, mithomycin C, mitramycin, methotrexate, docetaxel,etoposide (VP-16), paclitaxel, docetaxel, and adriamycin); andimmunosuppressants (e.g., cyclosporine A, tacrolimus). In someembodiments, the compounds, compositions and methods provided herein maybe in combination with histone deacetylase inhibitors (HDAC), aurorakinase inhibitors, demethylating agents (such as 5-AZA cytidine),immunotherapy with natural killer cells, IGF-IR antibodies, Ewingantigen antibodies, immunosuppressive drugs, and hydroxyurea. Examplesof histone deacetylase inhibitors include vorinostat, romidepsin,panobinostat, valproic acid, belinostat, mocetinostat, givinostat, andtrichostatin A. Examples of aurora kinase inhibitors include ZM447439,hesperadin, and VX-680. Examples of demethylating agents include5-azacytidine, 5-azadeoxycytidine, and procaine. Examples ofimmunosuppressive drugs include 6-mercaptopurine, and azathioprine.

Certain Kits

The compounds of the preferred embodiments can be provided to anadministering physician or other health care professional in the form ofa kit. The kit is a package which houses a container which contains thecompounds in a suitable pharmaceutical composition, and instructions foradministering the pharmaceutical composition to a subject. The kit canoptionally also contain one or more additional therapeutic agents, e.g.,chemotherapeutics currently employed for treating the sarcomas describedherein. For example, a kit containing one or more compositionscomprising compounds of the preferred embodiments in combination withone or more additional chemotherapeutic agents can be provided, orseparate pharmaceutical compositions containing an inhibitor of thepreferred embodiments and additional therapeutic agents can be provided.The kit can also contain separate doses of a compound of the preferredembodiments for serial or sequential administration. The kit canoptionally contain one or more diagnostic tools and instructions foruse. The kit can contain suitable delivery devices, e.g., syringes, andthe like, along with instructions for administering the inhibitor(s) andany other therapeutic agent. The kit can optionally contain instructionsfor storage, reconstitution (if applicable), and administration of anyor all therapeutic agents included. The kits can include a plurality ofcontainers reflecting the number of administrations to be given to asubject.

Certain Therapeutic Methods

Some embodiments provided herein relate to methods of treating theEwing's sarcoma family of tumors (ESFT). ESFT contains the unique fusionprotein EWS-FLI1. ESFT affects patients between the ages of 3 and 40years, with most cases occurring in the second decade. Although theembryologic cell type from which ESFT are derived is unknown, the tumoroften grows in close proximity to bone, but can occur as a soft-tissuemass. Over 40% of patients who present with localized tumors willdevelop recurrent disease and the majority of these will die from ESFT,while 75-80% of patients who present with metastatic ESFT will diewithin 5 years despite high-dose chemotherapy (Grier H E, Krailo M D,Tarbell N J, et al. Addition of ifosfamide and etoposide to standardchemotherapy for Ewing's sarcoma and primitive neuroectodermal tumor ofbone. N Engl J Med 2003; 348(8):694-701). These survival rates have notimproved for the past 20 years, even after dose-intensifyingchemotherapy. To improve survival and reduce therapy-related morbidity,novel targeted strategies for treating ESFT patients, as provided in thepreferred embodiments, can be employed.

ESFT are characterized by a translocation, occurring in 95% of tumors,between the central exons of the EWS gene (Ewing Sarcoma) located onchromosome 22 to the central exons of an ets family gene; either FLI1(Friend Leukemia Insertion) located on chromosome 11, t(11; 22), or ERGlocated on chromosome 21, t(21; 22). The EWS-FLI1 fusion transcriptencodes a 55 kDa protein (electrophoretic motility of approximately 68kD) with two primary domains. The EWS domain is a potent transcriptionalactivator, while the FLI1 domain contains a highly conserved ets DNAbinding domain (May W A, Lessnick S L, Braun B S, et al. The Ewing'ssarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptionalactivator and is a more powerful transforming gene than FLI-1. Mol CellBiol 1993; 13(12):7393-8); the resulting EWS-FLI1 fusion protein acts asan aberrant transcription factor. EWS-FLI1 transformation of mousefibroblasts requires both the EWS and FLI1 functional domains to beintact (May W A, Gishizky M L, Lessnick S L, et al. Ewing sarcoma 11; 22translocation produces a chimeric transcription factor that requires theDNA-binding domain encoded by FLI1 for transformation. Proc Natl AcadSci USA 1993; 90(12):5752-6).

EWS-FLI1 is an outstanding therapeutic target, in that it is expressedonly in tumor cells and is required to maintain the growth of ESFT celllines. Reduced expression levels of EWS-FLI1 using either antisenseoligodeoxynucleotides (ODN) (Toretsky J A, Connell Y, Neckers L, Bhat NK. Inhibition of EWS-FLI-1 fusion protein with antisenseoligodeoxynucleotides. J Neurooncol 1997; 31(1-2): 9-16; Tanaka K,Iwakuma T, Harimaya K, Sato H, Iwamoto Y. EWS-Fli1 antisenseoligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma andprimitive neuroectodermal tumor cells. J Clin Invest 1997; 99(2):239-47)or small interfering RNAs (siRNA) (Ouchida M, Ohno T, Fujimura Y, Rao VN, Reddy E S. Loss of tumorigenicity of Ewing's sarcoma cells expressingantisense RNA to EWS-fusion transcripts. Oncogene 1995; 11(6):1049-54;Maksimenko A, Malvy C, Lambert G, et al. Oligonucleotides targetedagainst a junction oncogene are made efficient by nanotechnologies.Pharm Res 2003; 20(10):1565-7; Kovar H, Aryee D N, Jug G, et al.EWS/FLI-1 antagonists induce growth inhibition of Ewing tumor cells invitro. Cell Growth Differ 1996; 7(4):429-37) cause decreasedproliferation of ESFT cell lines and regression of tumors in nude mice.Recent advances in nanotechnology have improved the delivery andcontrolled release of siRNA, yet neither antisense ODN nor siRNAreduction of EWS-FLI1 in humans is possible with current technologies(Maksimenko A, Malvy C, Lambert G, et al. Oligonucleotides targetedagainst a junction oncogene are made efficient by nanotechnologies.Pharm Res 2003; 20(10):1565-7; Lambert G, Bertrand J R, Fattal E, et al.EWS fli-1 antisense nanocapsules inhibits Ewing sarcoma-related tumor inmice. Biochem Biophys Res Commun 2000; 279(2):401-6). One interestingapproach to EWS-FLI1 targeting used comparative expression between siRNAreduced EWS-FLI1 and a library of small molecules, which led to acurrent clinical trial with Ara-C (Stegmaier K, Wong J S, Ross K N, etal. Signature-based small molecule screening identifies cytosinearabinoside as an EWS/FLI modulator in Ewing sarcoma. PLoS medicine2007; 4(4):e122). This method of identifying Ara-C also indicateddoxorubicin and puromycin would reduce EWS-FLI1 levels. Doxorubicin iscurrently used as standard therapy for ESFT patients and yet, survivalis far from acceptable (Grier H E, Krailo M D, Tarbell N J, et al.Addition of ifosfamide and etoposide to standard chemotherapy forEwing's sarcoma and primitive neuroectodermal tumor of bone. N Engl JMed 2003; 348(8):694-701). The use of Ara-C in ESFT patients iscurrently being evaluated in a Phase II trial. While it is hoped thatthis represents a needed clinical breakthrough, it certainlydemonstrates the importance of small molecule targeting of EWS-FLI1. Thepreferred embodiments provide small molecule protein-protein interactioninhibitors (SMPPII) that disrupt EWS-FLI1 from critical proteinpartners, thereby achieving tumor specificity and more precise targetingof EWS-FLI1.

There is sufficient evidence to conclude that EWS-FLI1 fusion proteinfunctions differently than either untranslocated EWS or FLI1 (May W A,Gishizky M L, Lessnick S L, et al. Ewing sarcoma 11; 22 translocationproduces a chimeric transcription factor that requires the DNA-bindingdomain encoded by FLI1 for transformation. Proc Natl Acad Sci USA 1993;90(12):5752-6). Changes in gene expression profiles ofEWS-FLI1-expressing cell lines (Braun B S, Frieden R, Lessnick S L, MayW A, Denny C T. Identification of target genes for the Ewing's sarcomaEWS/FLI fusion protein by representational difference analysis. Mol CellBiol 1995; 15(8):4623-30) or tumor cells taken from ESFT patients,compared to tumors lacking EWS-FLI1 expression, indicate that EWS-FLI1may play a role in transcriptional regulation (Khan J, Wei J S, RingnerM, et al. Classification and diagnostic prediction of cancers using geneexpression profiling and artificial neural networks. Nat Med 2001;7(6):673-9; Baer C, Nees M, Breit S, et al. Profiling and functionalannotation of mRNA gene expression in pediatric rhabdomyosarcoma andEwing's sarcoma. Int J Cancer 2004; 110(5):687-94). While a clearpicture of the mechanism of EWS-FLI1-regulated gene expression has yetto emerge, this activity is likely the result of direct or secondaryinteractions between EWS-FLI1 and regulators of RNA synthesis andsplicing (Uren A, Toretsky J A. Ewing's Sarcoma Oncoprotein EWS-FLI1:the Perfect Target without a Therapeutic Agent. Future Onc 2005;1(4):521-8).

EWS-FLI1 is a great therapeutic target since it is only expressed intumor cells; however, the ability to target this tumor-specific oncogenehas previously not been successful. One of the challenges towards smallmolecule development is that EWS-FLI1 lacks any know enzymatic domains,and enzyme domains have been thought to be critical for targetedtherapeutics. In addition, EWS-FLI1 is a disordered protein, indicatingthat it does not exhibit a rigid structure that can be used forstructure based drug design (Uren A, Tcherkasskaya O, Toretsky J A.Recombinant EWS-FLI1 oncoprotein activates transcription. Biochemistry2004; 43(42):13579-89). In fact, the disordered nature of EWS-FLI1 iscritical for its transcriptional regulation (Ng K P, Potikyan G, SaveneR O, Denny C T, Uversky V N, Lee K A. Multiple aromatic side chainswithin a disordered structure are critical for transcription andtransforming activity of EWS family oncoproteins. Proc Natl Acad Sci USA2007; 104(2):479-84). Disordered proteins are considered as moreattractive targets for small molecule protein-protein interactioninhibitors specifically because of their biochemical disorderedproperties (Cheng Y, LeGall T, Oldfield C J, et al. Rational drug designvia intrinsically disordered protein. Trends Biotechnol 2006;24(10):435-42).

EWS-FLI1 binds RNA helicase A in vitro and in vivo. It is believed thatprotein-protein interactions of EWS-FLI1 may contribute to its oncogenicpotential; therefore, novel proteins have been sought that directlyinteract with and functionally modulate EWS-FLI1. Recombinant EWS-FLI1that is transcriptionally active (Uren A, Tcherkasskaya O, Toretsky J A.Recombinant EWS-FLI1 oncoprotein activates transcription. Biochemistry2004; 43(42):13579-89) was used as a target for screening a commercialpeptide phage display library. Twenty-eight novel peptides thatdifferentially bind to EWS-FLI1 were identified from phage sequencing. ANational Center for Biotechnology Information database search for humanproteins homologous to these peptides identified a peptide that washomologous to aa 823-832 of the human RNA helicase A, (RHA, gene bankaccession number A47363) (Toretsky J A, Erkizan V, Levenson A, et al.Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res2006; 66(11):5574-81).

RHA, a member of the highly conserved DEXD/H box helicase family ofproteins, is an integral, multifunctional member of the humantranscriptome (Zhang S, Grosse F. Multiple functions of nuclear DNAhelicase II (RNA helicase A) in nucleic acid metabolism. Acta BiochimBiophys Sin (Shanghai) 2004; 36(3):177-83; von Hippel P H, Delagoutte E.A general model for nucleic acid helicases and their “coupling” withinmacromolecular machines. Cell 2001; 104(2):177-90). These proteins areinvolved in diverse functions in a variety of organisms, from archaea,eubacteria, lower and higher eukaryotes and a number of viruses,including the positive-sense RNA viruses of the Flavivirus family. RHAis a transcriptional coactivator for NF-κB, and has been shown to formcomplexes with Creb-binding protein (CBP) (Nakajima T, Uchida C,Anderson S F, et al. RNA helicase A mediates association of CBP with RNApolymerase II. Cell 1997; 90(6):1107-12), RNA Polymerase II (Nakajima T,Uchida C, Anderson S F, et al. RNA helicase A mediates association ofCBP with RNA polymerase II. Cell 1997; 90(6):1107-12), the breast cancertumor suppressor BRCA1 (Anderson S F, Schlegel B P, Nakajima T, Wolpin ES, Parvin J D. BRCA1 protein is linked to the RNA polymerase IIholoenzyme complex via RNA helicase A. Nat Genet 1998; 19(3):254-6),and, most recently, EWS-FLI1 (Toretsky J A, Erkizan V, Levenson A, etal. Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A. CancerRes 2006; 66(11):5574-81). EWS-FLI1 binds to a region of RHA that isunique and not known as a binding site for any of the other RHA bindingpartners (Toretsky J A, Erkizan V, Levenson A, et al. OncoproteinEWS-FLI1 activity is enhanced by RNA helicase A. Cancer Res 2006;66(11):5574-81). RHA expression enhanced EWS-FLI1 mediatedanchorage-independent colony formation, while an inactivating mutationof RHA prevented colony formation (Toretsky J A, Erkizan V, Levenson A,et al. Oncoprotein EWS-FLI1 activity is enhanced by RNA helicase A.Cancer Res 2006; 66(11):5574-81). This structural and functioninteraction is the basis for the therapeutic agents of preferredembodiments.

Despite the importance of transcription in tumorigenesis, the role ofhelicases in this process has not been well-studied. RHA is an integralmember of the human transcriptome with diverse functions (Zhang S,Grosse F. Multiple functions of nuclear DNA helicase II (RNA helicase A)in nucleic acid metabolism. Acta Biochim Biophys Sin (Shanghai) 2004;36(3):177-83; von Hippel P H, Delagoutte E. A general model for nucleicacid helicases and their “coupling” within macromolecular machines. Cell2001; 104(2):177-90). Our recently published data show that RHAinteracts with the multifunctional EWS-FLI1 oncoprotein (Toretsky J A,Erkizan V, Levenson A, et al. Oncoprotein EWS-FLI1 activity is enhancedby RNA helicase A. Cancer Res 2006; 66(11):5574-81). This interactioncould account for the observed ability of EWS-FLI1 to function in bothtranscription initiation and post-transcriptional RNA modification. RNAhelicases are also known to bind and act as a bridge for some of thesame factors that have been identified as binding partners for EWS-FLI1,including the splicing factor U1C (Chen J Y, Stands L, Staley J P,Jackups R R, Jr., Latus L J, Chang T H. Specific alterations of U1-Cprotein or U1 small nuclear RNA can eliminate the requirement of Prp28p,an essential DEAD box splicing factor. Mol Cell 2001; 7(1):227-32; KnoopL L, Baker S J. The splicing factor U1C represses EWS/FLI-mediatedtransactivation. J Biol Chem 2000; 275(32):24865-71), Creb-bindingprotein (CBP) (Nakajima T, Uchida C, Anderson S F, et al. RNA helicase Amediates association of CBP with RNA polymerase II. Cell 1997;90(6):1107-12) and RNA Polymerase II (Nakajima T, Uchida C, Anderson SF, et al. RNA helicase A mediates association of CBP with RNA polymeraseII. Cell 1997; 90(6):1107-12). RHA may perform a similar function forEWS-FLI1 and RNA Pol II, acting in the recruitment of key processingproteins. RHA may also contribute to ESFT oncogenesis by maintainingEWS-FLI1 as part of a large transcriptional complex whose functionrelies on the ATPase activity of RHA as an energy source. Finally,helicases, like RHA, can stabilize mRNA species (Iost I, Dreyfus M.mRNAs can be stabilized by DEAD-box proteins. Nature 1994;372(6502):193-6). The stabilization and metabolism of EWS-FLI1transcribed mRNA by RHA may augment the oncogenic nature of EWS-FLI1.

While EWS-FLI1 is quite specific to ESFT cells, EWS and RHA areubiquitously expressed. The region between EWS-FLI1 and RHA are targetedby molecular therapeutics that may have specificity; since EWS-FLI1 isexpressed only in tumors and the interaction points with RHA may beunique. Therapeutic agents, namely, small molecule protein-proteininteraction inhibitors, are provided herein to inhibit EWS-FLI1function.

Most translocation-fusion protein sarcomas portend a poor prognosis,including ESFT. The chromosomal translocation t(11; 22), leading to theunique and critical fusion protein EWS-FLI1, is a perfect cancer target.Many other sarcomas share similar translocation variants (Table 2. fromHelman L J, Meltzer P. Mechanisms of sarcoma development. Nat Rev Cancer2003; 3(9):685-94).

EWS-FLI1 translocations have been reported in solidpseudopapillaryneoplasms of the pancreas (Maitra A., et al., Detectionof t(11; 22)(q24; q12) translocation and EWS-FLI-1 fusion transcript ina case of solid pseudopapillary tumor of the pancreas. Pediatr DevPathol 2000; 3:603-605), however the role of EWS-FLI1 in all solidpseudopaillary neoplasms remains to be resolved (Katharina Tiemann etal., Solid pseudopapillary neoplasms of the pancreas are associated withFLI-1 expression, but not with EWS/FLI-1 translocation).

EWS or FLI1 homologues are partners in translocations that occur in awide range of sarcomas and leukemias. EWS, or its homologue TLS or FUS,is involved in chromosomal translocations of clear cell sarcoma, myxoidliposarcoma, desmoplastic small round cell tumor, chondrosarcoma andacute myeloid leukemia. FLI1 belongs to the ets family of genes. TheFLI1 homologue ERG is translocated in approximately 10% of Ewing'ssarcomas and 20% of acute myeloid leukemias. This suggests that EWS-FLI1can serve as model system that might impact upon a family of diseases(related by translocation partners) that affect a large number ofpatients (Uren A., Tcherkasskaya O. and Toretsky J. A. RecombinantEWS-FLI1 oncoprotein activates transcription. Biochemistry 43(42)13579-89 (2004)).

ERG is also translocated in prostate cancer, where the TMPRSS2:ERGfusion suggests a distinct molecular subtype that may define risk fordisease progression (F. Demichelis et al., TMPRSS2:ERG gene fusionassociated with lethal cancer in a watchful waiting cohort. Oncogene(2007)26, 4596-4599). Other diseases where translocations of EWS or FLI1family members have been observed include congenital fibrosarcoma andcellular mesoblastic nephroma where the ets family member ETV6 isjuxtaposed with NTRK3. Other translocation gene fusions include chronicmyeloid leukemia that leads to expression of the BCR-ABL fusion protein,and synovial sarcoma where the SYT gene from chromosome 18 is juxtaposedwith either SSX1 or SSX2 from the X chromosome (Aykut Uren and JeffreyA. Toretsky, Pediatric malignancies provide unique cancer therapytargets. Curr Opin Pediatr 17:14-19 (2005)).

Therefore, the therapeutic agents of the preferred embodiments havepotential for application in many other tumors. More broadly, some ofthe most difficult leukemias also have translocation-generated fusionproteins involving the mixed-lineage leukemia gene (MLL, 11q23), and ourwork could serve as a paradigm for a very treatment-resistant group ofcancers (Pui C H, Chessells J M, Camitta B, et al. Clinicalheterogeneity in childhood acute lymphoblastic leukemia with 11q23rearrangements. Leukemia 2003; 17(4):700-6.). Thus embodiments includecancers where translocations have occurred. Translocation fusion genesare listed in Table 1.

TABLE 1 Translocation Genes Type of fusion gene Ewing's sarcoma t(11;22)(q24; q12) EWSR1-FLI1 Transcription factor t(21; 22)(q22; q12)EWSR1-ERG Transcription factor t(7; 22)(p22; q12) EWSR1-ETV1Transcription factor t(17; 22)(q21; q12) EWSR1-ETV4 Transcription factort(2; 22)(q33; q12) EWSR1-FEV Transcription factor Clear-cell sarcomat(12; 22)(q13; q12) EWSR1-ATF1 Transcription factor Desmoplastic smallround-cell tumor t(11; 22)(p13: q12) EWSR1-WT1 Transcription factorMyxoid chondrosarcoma t(9; 22)(q22-31; q11-12) EWSR1-NR4A3 Transcriptionfactor Myxoid liposarcoma t(12; 16)(q13; p11) FUS-DDIT3 Transcriptionfactor t(12; 22)(q13; q12) EWSR1-DDIT3 Transcription factor Alveolarrhabdomyosarcoma t(2; 13)(q35; q14) PAX3-FOXO1A Transcription factort(1; 13)(p36; q14) PAX7-FOXO1A Transcription factor Synovial sarcomat(X; 18)(p11; q11) SYT-SSX Transcription factor Dermatofibrosarcomaprotuberans t(17; 22)(q22; q13) COL1A1-PDGFB Growth factor Congenitalfibrosarcoma t(12; 15)(p13; q25) ETV6-NTRK3 Transcription-factorreceptor Inflammatory myofibroblastic tumor 2p23 rearrangementsTMP3-ALK; TMP4- Growth-factor ALK receptor Alveolar soft-part sarcomat(X; 17)(p11.2; q25) ASPL-TFE3 Transcription factor

Certain Indications

Certain compounds, compositions and methods provided herein can be usedto treat a number of disorders such as a tumor comprising atranslocation gene fusion, Ewing's sarcoma, clear cell sarcoma, myxoidliposarcoma, desmoplastic small round-cell tumor, myxoid chondrosarcoma,acute myeloid leukemia, congenital fibrosarcoma, prostate cancer, breastcancer, and pancreatic cancer. In some embodiments, the cancer is lungadenocarcinoma, or glioblastoma multiforme. In some embodiments, thecancer comprises a translocation comprising an ETS gene selected fromthe group consisting of FLI1, ETV1, ETV4, ERG, ETS1, and ETS2.

EXAMPLES

The following examples, including experiments and results achieved, areprovided for illustrative purposes only and are not to be construed aslimiting the present invention. Where chemical structures depict atomshaving an unfilled valency, it is to be understood that the valency issatisfied with one or more hydrogen atoms.

Example 1—Synthesis of 4,7 Dichloroisatin Analogs

An appropriate acetophenone and 4, 7-dichloroisatin were condensed inthe presence of a catalytic amount of diethylamine to prepare thedesired compound in quantitative yield. Example compounds: R¹=4′-CN(PT-1-11); 2′-OCH₃ (PT-1-12); 3′-OCH₃ (PT-1-18); 2′,4′-OCH₃ (PT-1-19);2′,3′-OCH₃ (PT-1-20); 3′,4′OCH₃ (PT-1-21); 3′,5′OCH₃ (PT-1-22);2′,3′,4′,-OCH₃ (PT-1-23); 3′,4′,5′-OCH₃ (PT-1-13); 4′-OC₂H₅ (PT-1-14);4′-CF₃ (PT-1-15); 4′-OCF₃ (PT-1-16); 4′-N(CH₃)₂ (PT-1-17); 4′-OPh(PT-1-60); 4′-SCH₃ (PT-1-67); and 4′-C(CH₃)₂ (PT-1-67).

Example 2—Synthesis of Dehydrated 4,7 Dichloroisatin Analogs

A solution of 4,7-dichloroisatin in 96% H₂SO₄ was stirred at roomtemperature to yield the reduced analogs. Example compounds: R2=4′-OCH3(PT-1-33); 2′,4′-OCH3 (PT-1-39); 2′,3′,4′,-OCH3 (PT-1-41); 4′-OC2H5(PT-1-43); and 4′-N(CH3)2 (PT-1-38).

Example 3—Synthesis of Reduced 4,7 Dichloroisatin Analogs

Example 4—Synthesis of Reduced 4,7 Dichloroisatin Pyridine Derivatives

Example 5—Biological Activity of Certain Compounds

Compounds provided in Table 2 were prepared using methods similar tothose described herein. The structures and IC₅₀ activities of particularcompounds in PANC1 (a human pancreatic carcinoma), TC32 (human ESFT cellline), and TC71 (human ESFT cell line) cells are summarized in Table 2.

TABLE 2 IC₅₀ (μM) Example Structure PANC 1 TC32 TC71 YK-4-275

11 40 23.95 YK-4-279

19.98; 33.96 0.9395; 0.7657 0.9178; 1.426 YK-4-280

40 12.11 30.08 YK-4-281

40 7.218 29.61 YK-4-283

12.66 8.911 25.96 YK-4-284

40 40 40 YK-4-285

40 40 40 YK-4-286

40 4.631 9.149 YK-4-287

12.6 6.32 15.82 YK-4-288

40 3.002 9.345 YK-4-289

40 40 40 PT-1-11

40 10.34 12.28 PT-1-14

11.11 2.698 3.568 PT-1-15

10.91 2.952 6.941 PT-1-17

40; 40 0.2589; 0.2836 0.4008; 0.2945 PT-1-18

40 40 40 PT-1-19

22.94 2.609 2.819 PT-1-22

40 8.988 40 PT-1-23

40 2.698 4.422 PT-1-38

15.5; 40 0.2908; 0.3833 40; 0.5682 PT-1-39

5.413; 6.763 1.052; 1.664 1.806; 2.318 PT-1-41

2.855; 5.158 1.194; 1.611 2.142; 1.599 PT-1-43

10.98 1.409 5.655 PT-1-53

2.202 40 4.08 PT-1-54

2.127; 40 1.498; 2.57 1.362; 2.202 PT-1-60

40 40 40 PT-1-64 40 32.8 40 PT-1-67

28.1; 40 0.9822; 1.203 0.9086; 1.409 PT-1-69

40 40 40 PT-1-267

40 40 40 PT-1-271

40 40 40 PT-1-275

40 40 40 PT-2-39

40 40 40 PT-2-52

40 40 40 PT-2-56

40 12.36 40 PT-2-59

40 40 40 PT-2-64

40 40 40 PT-2-69

40; 40 2.178; 2.305 0.7145; 2.341 PT-2-71

40 40 40 YK-4-276

40 40 40 YK-4-277

40 40 40 YK-4-278

40 40 40 YK-4-282

40 40 40 PT-1-12

40 40 40 PT-1-13

40 40 40 PT-1-16

40 40 40 PT-1-20

40 40 40 PT-1-21

40 40 40 PT-1-33

40 1.035 1.636 PT-2-37

40 40 40 PT-2-78

40 40 40 PT-2-79

11.19 12.13 16.98 PT-2-47

PT-2-39

PT-2-99

PT-2-94

PT-2-84

PT-2-89

Example 6—Growth Inhibition of EWS-FLI1 Cells with Substituted Analogs

The effects of the YK-4-279 analogs on the ESFT cells were tested bydetermining their growth inhibition. The IC50 of the lead compound was900 nM for cells growing in monolayer. Growth inhibition of ESFT cellswas measured for various concentrations of particular compounds. Growthinhibition of TC71 and TC32 cells was measured for variousconcentrations of YK-4-279 and PT-1-33 (FIG. 3A). Growth inhibition ofTC71 cells was measured for various concentrations of YK-4-279, PT-1-33,and PT-1-55 (FIG. 3B). Growth inhibition of TC71 cells was measured forvarious concentrations of YK-4-279 and PT-1-123 (FIG. 3C). Some of theanalogs had similar activity to YK-4-279. The dehydrated analogs and thealcohol analogs showed a similar activity against ESFT cells (FIG. 3A).Modifications of the ketone did not improve the activity of compounds(FIG. 3B and FIG. 3C).

Example 7—Apoptosis of EWS-FLI1 Cells

Immunoblots were prepared from protein lysates from TC32 cells treatedwith YK-4-279 and co-precipitated with RHA, EWS-FLI1 or total protein(FIG. 4). YK-4-279 did not directly affect the level of EWS-FLI1 or RHAbut did disrupt their interactions. The disruption of the interaction ofRHA with EWS-FLI1 presents an avenue for the development of a class ofsmall molecules as potential therapeutics against the Ewing's familysarcoma tumors. While YK-4-279 disrupted the protein-proteininteraction, PT-1-17 appeared to be more potent in the TC71 cells.Dehydrated analogs of YK-4-279 did not significantly increase thepotency of the compounds.

Example 8—Disruption of EWS-FLI1/RHA Binding

The activity of candidate small molecules to disrupt binding betweenEWS-FLI1 and the His-tagged RHA protein, His-Tag RHA (647-1075), wasscreened in an ELISA assay. Briefly, candidate agents were incubatedwith RHA on plates coated with EWS-FLI1. After washing the plates, theamount of RHA that remained bound to the plates was determined using aprimary anti-RHA antibody, and a secondary signal antibody.

Wells in a 96-well plate were incubated with 100 μl/well 20 nM EWS-FLI1protein solution (1M imidazole, 20 mM Tris, 500 mM NaCl) overnight at 4°C. Plates were washed with PBS, blocked with 150 μl/well 4% BSA for atleast 2 h at room temperature, and then washed again with ELISA washsolution (PBS+0.1% T20, 200 μl/well). Plates were incubated for 1 hourat room temperature with 100 μl/well candidate agent in PBS (10 μM or 50μM final), or DMSO control. Plates were incubated overnight at 4° C.with 100 μl/well 20 nM His-RHA protein solution (0.5 M imidazole, 125 mMNaCl, 20 mM Tris), and then washed with ELISA wash solution (PBS+0.1%T20, 200 μl/well). RHA bound to the plates was detected by incubatingplates for 1 hour at room temperature with 100 μl/well primary anti-RHAantibody (1:1000 goat Anti-DHX9/EB09297, Everest), and then washing withELISA wash solution (PBS+0.1% T20, 200 μl/well). Primary antibody wasdetected by incubating plates for 1 hour at room temperature with 100μl/well secondary anti-goat antibody (1:500 donkey anti-goat IgG-HRP:sc-2020), and then washing with ELISA wash solution (PBS+0.1% T20, 200μl/well). A horseradish peroxidase assay kit was used to determine theamount of secondary anti-goat antibody in each well (Bio-Rad—TMBPeroxidase EIA Substrate Kit #172-1066), with plates read at 450 nm. Arelatively lower optical density indicating lower amounts of HRPindicate a candidate agent with increased inhibitory activity forEWS-FLI1-RHA binding. The results are summarized in FIGS. 5A-5G. FIG. 5Asummarizes results for the following candidate molecules: YK-4-275,YK-4-285, PT-1-12, PT-1-18, PT-1-19, PT-1-20, PT-1-21, PT-1-22, PT-1-23,PT-1-175. FIG. 5B summarizes results for the following candidatemolecules: PT-2-84, PT-2-59, PT-1-17, PT-2-71, PT-2-89, PT-1-123,PT-1-15, PT-1-60, PT-1-67, PT-1-69. FIG. 5C summarizes results for thefollowing candidate molecules: YK-4-285, YK-4-286, PT-1-33, PT-1-38,PT-1-271, PT-1-52, PT-1-56, PT-1-64, PT-2-94, PT-1-267). FIG. 5Dsummarizes results for the following candidate molecules: YK-4-282,YK-4-287, YK-4-2 80, YK-4-289, YK-4-288, YK-4-278, YK-4-276, YK-4-283,YK-4-277, YK-4-281 FIG. 5E summarizes results for the followingcandidate molecules: PT-1-54, YK-4-279 (S), YK-4-279 (R), PT-1-55,PT-2-75, PT-2-39, PT-2-79, PT-1-16, PT-1-13, PT-2-64. FIG. 5F summarizesresults for the following candidate molecules: YK-4-284, PT-1-14,PT-1-39, PT-1-41, PT-1-43, PT-1-53, PT-2-56, PT-2-52, PT-1-61, PT-1-183.FIG. 5G summarizes results for the following candidate molecules:PT-1-275, PT-2-69, PT-2-99, YK-4-288, PT-1-19, PT-1-20, PT-1-69,PT-2-89, PT-1-17, PT-2-94.

Example 9-Disruption of EWS-FLI1 Transcription Factor Activity

The activity of candidate small molecules to disrupt EWS-FLI1transcription factor activity was screened using a luciferase assay inwhich EWS-FLI1 binding to the NROB1 promoter increases luciferaseexpression. Briefly, cells were transfected with a vector containing theNROB1 promoter driving luciferase expression, and an EWS-FLI1 expressionvector. Transfected cells were treated with various concentrations of acandidate agent, and any change in the relative level of luciferaseexpression was determined. COST cells were plated in 96-well plates andtransfected with pciNEO/EF vector and pGL3-NROB1. Controls includedtransfections with each vector only. Transfected cells were treated withvarious concentrations of a candidate agent, and treated cells wereassays for luciferase activity. Decreased luciferase activity indicatesa candidate agent with inhibitory activity in EWS-FLI1 acting as atranscription factor, promoting transcription of luciferase. FIG. 6A andFIG. 6B show general trends for relative luciferase activity for variousconcentrations of candidate agents. FIGS. 7A-7I show inhibitory activityfor various concentrations of candidate agents.

Example 10-Treating Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a very well annotated tumor from theperspective of its genetics that have led to molecular segregation intoclassic, proneural, neural, and mesenchymal categories (Purow B W,Schiff D. Glioblastoma genetics: in rapid flux. Discov Med. 2010February; 9(45):125-31. PubMed PMID: 20193638. Pubmed Central PMCID:3365574). The genetic alterations that categorize GBM includeconstitutive activation of signaling pathways, loss of tumorsuppressors, mutations in metabolic pathways, abnormal DNA repair, andloss of mitotic regulators (Suzuki E, Williams S, Sato S, Gilkeson G,Watson D K, Zhang X K. The transcription factor Fli-1 regulatesmonocyte, macrophage and dendritic cell development in mice. Immunology.2013 July; 139(3):318-27. PubMed PMID: 23320737. Pubmed Central PMCID:3701178; Chow L M, Endersby R, Zhu X, Rankin S, Qu C, Zhang J, et al.Cooperativity within and among Pten, p53, and Rb pathways induceshigh-grade astrocytoma in adult brain. Cancer Cell. 2011 Mar. 8;19(3):305-16. PubMed PMID: 21397855. Pubmed Central PMCID: 3060664;Solomon D A, Kim T, Diaz-Martinez L A, Fair J, Elkahloun A G, Harris BT, et al. Mutational inactivation of STAG2 causes aneuploidy in humancancer. Science. 2011 Aug. 19; 333(6045):1039-43. PubMed PMID: 21852505.Epub 2011/08/20. eng). Even within these categories, GBM is recognizedas a tumor with significant intratumoral heterogeneity (Garraway L A,Lander E S. Lessons from the cancer genome. Cell. 2013 Mar. 28;153(1):17-37. PubMed PMID: 23540688; Nabilsi N H, Deleyrolle L P, DarstR P, Riva A, Reynolds B A, Kladde M P. Multiplex mapping of chromatinaccessibility and DNA methylation within targeted single moleculesidentifies epigenetic heterogeneity in neural stem cells andglioblastoma. Genome Res. 2013 Oct. 8. PubMed PMID: 24105770). Despitethis extraordinary variability in genetics, little attention has beenfocused on transcriptional regulators. One reason transcription factorshave been less well studied in GBM may be the absence of effective smallmolecule inhibitors. The exception to this is p53, whose wild-typefunction can be sustained with the small molecule protein interactioninhibitor Nutlin-3 (Vassilev L T. p53 Activation by small molecules:application in oncology. J Med Chem. 2005 Jul. 14; 48(14):4491-9. PubMedPMID: 15999986).

Despite the genetic diversity, many clinical trials have been completedthat evaluate targeted and non-targeted therapeutics for GBM. Despiteall of these trials, including radiation therapy and molecularly guidedsurgical resection, progress towards effective, long-term GBM therapyhas been unsuccessful for the vast majority of patients (Yin A A, ChengJ X, Zhang X, Liu B L. The treatment of glioblastomas: A systematicupdate on clinical Phase III trials. Crit Rev Oncol Hematol. 2013September; 87(3):265-82. PubMed PMID: 23453191). A most recent VEGFRsmall molecule inhibitor plus temozolomide phase III trial also showedno improvement over standard of care temozolomide plus radiation therapy(Batchelor T T, Mulholland P, Neyns B, Nabors L B, Campone M, Wick A, etal. Phase III Randomized Trial Comparing the Efficacy of Cediranib AsMonotherapy, and in Combination With Lomustine, Versus Lomustine Alonein Patients With Recurrent Glioblastoma. J Clin Oncol. 2013 Sep. 10;31(26):3212-8. PubMed PMID: 23940216). A significant challenge to anyGBM therapy is overcoming the blood-brain barrier (BBB), and this hasimpacted many potential targeted therapies (Juratli T A, Schackert G,Krex D. Current status of local therapy in malignant gliomas—a clinicalreview of three selected approaches. Pharmacol Ther. 2013 September;139(3):341-58. PubMed PMID: 23694764).

The use of microRNA (miRNA) both to understand the biology of GBM anddevelop novel therapies led to a novel discovery that diacylglycerolkinase alpha may be a potential target, and small molecule optimizationsare currently underway for these inhibitors (Dominguez C L, Floyd D H,Xiao A, Mullins G R, Kefas B A, Xin W, et al. Diacylglycerol kinasealpha is a critical signaling node and novel therapeutic target inglioblastoma and other cancers. Cancer Discov. 2013 July; 3(7):782-97.PubMed PMID: 23558954. Pubmed Central PMCID: 3710531). In addition, themiRNA have been used to create bioinformatics models that do suggest anetwork of transcriptional regulation is clearly important for GBMoncogenesis (Sun J, Gong X, Purow B, Zhao Z. Uncovering MicroRNA andTranscription Factor Mediated Regulatory Networks in Glioblastoma. PLoScomputational biology. 2012; 8(7):e1002488. PubMed PMID: 22829753.Pubmed Central PMCID: 3400583).

Friend Leukemia Insertion-1 (FLI1) is a Putative Novel GBM Target.

Transcription factors are the focal driving oncogene in many cancers,yet have been considered ‘undruggable’ since they lack enzymaticactivity. To date, despite the TCGA database for GBM, the therapeutictargeting of critical transcriptional nodes has not occurred. The etsfamily transcription factor FLI1 is expressed in GBM based upon queryingthe TCGA database (FIG. 8). Early ETS-1 studies correlated ETS-1expression with malignant potential in human astrocytic tumors (KitangeG, Kishikawa M, Nakayama T, Naito S, Iseki M, Shibata S. Expression ofthe Ets-1 proto-oncogene correlates with malignant potential in humanastrocytic tumors. Mod Pathol. 1999 June; 12(6):618-26. PubMed PMID:10392639). In addition, studies showed that ETS-1 may drive angiogenesisin astrocytic tumors (Valter M M, Hugel A, Huang H J, Cavenee W K,Wiestler O D, Pietsch T, et al. Expression of the Ets-1 transcriptionfactor in human astrocytomas is associated with Fms-like tyrosinekinase-1 (Flt-1)/vascular endothelial growth factor receptor-1 synthesisand neoangiogenesis. Cancer Res. 1999 Nov. 1; 59(21):5608-14. PubMedPMID: 10554042). Many studies suggest a significant role for ets familyELK members in GBM transcription and overall biology (Day B W, StringerB W, Spanevello M D, Charmsaz S, Jamieson P R, Ensbey K S, et al. ELK4neutralization sensitizes glioblastoma to apoptosis throughdownregulation of the anti-apoptotic protein Mcl-1. Neuro Oncol. 2011November; 13(11):1202-12. PubMed PMID: 21846680. Pubmed Central PMCID:3199151; Shukla A A, Jain M, Chauhan S S. Ets-1/Elk-1 is a criticalmediator of dipeptidyl-peptidase III transcription in human glioblastomacells. Febs J. 2010 April; 277(8):1861-75. PubMed PMID: 20236318; Uht RM, Amos S, Martin P M, Riggan A E, Hussaini I M. The protein kinaseC-eta isoform induces proliferation in glioblastoma cell lines throughan ERK/Elk-1 pathway. Oncogene. 2007 May 3; 26(20):2885-93. PubMed PMID:17146445). While one immunohistochemical study did not find FLI1expression in GBM, however, there are significant challenges in antibodyselection and antigen retrieval that may have impacted on these negativeresults (Mhawech-Fauceglia P, Herrmann F R, Bshara W, Odunsi K,Terracciano L, Sauter G, et al. Friend leukaemia integration-1expression in malignant and benign tumours: a multiple tumour tissuemicroarray analysis using polyclonal antibody. J Clin Pathol. 2007 June;60(6):694-700. PubMed PMID: 16917000. Pubmed Central PMCID: 195505).

Using the cBioPortal for cancer genomics website interface, a subset ofets family members involved in cancer was evaluated. These alterationsinclude amplifications (solid red bar), mutations (small green square),and mRNA upregulation (open red bars). See FIG. 8.

FLI1 targeting in GBM is further supported based upon itstranscriptional activation of MDM2 (Truong A H, Cervi D, Lee J,Ben-David Y. Direct transcriptional regulation of MDM2 by Fli-1.Oncogene. 2005 Feb. 3; 24(6):962-9. PubMed PMID: 15592502). In thiscase, high MDM2 would cause degradation of p53, leading to loss of a keytumor suppressor protein. Of note, there is a loose correlation amongthe seven GBM cell lines between those with high FLI1 and high MDM2(Table 3 and FIG. 10). In hematopoietic development, FLI1 is clearly animportant protein, as noted by multiple immune defects when protein iseliminated by homologous recombination (Suzuki E, Williams S, Sato S,Gilkeson G, Watson D K, Zhang X K. The transcription factor Fli-1regulates monocyte, macrophage and dendritic cell development in mice.Immunology. 2013 July; 139(3):318-27. PubMed PMID: 23320737. PubmedCentral PMCID: 3701178; Kruse E A, Loughran S J, Baldwin T M, JosefssonE C, Ellis S, Watson D K, et al. Dual requirement for the ETStranscription factors Fli-1 and Erg in hematopoietic stem cells and themegakaryocyte lineage. Proc Natl Acad Sci USA. 2009 Aug. 18;106(33):13814-9. PubMed PMID: 19666492. Pubmed Central PMCID: 2728977;Liu F, Walmsley M, Rodaway A, Patient R. Fli1 acts at the top of thetranscriptional network driving blood and endothelial development. CurrBiol. 2008 Aug. 26; 18(16):1234-40. PubMed PMID: 18718762). While FLI1is critical from embryogenesis, it is not likely to be critical inmature tissues since its expression is limited to subsets of immunecells and endothelium (Watson D K, Smyth F E, Thompson D M, Cheng J Q,Testa J R, Papas T S, et al. The ERGB/Fli-1 gene: isolation andcharacterization of a new member of the family of human ETStranscription factors. Cell Growth Differ. 1992 October; 3(10):705-13.PubMed PMID: 1445800; Truong A H, Ben-David Y. The role of Fli-1 innormal cell function and malignant transformation. Oncogene. 2000 Dec.18; 19(55):6482-9. PubMed PMID: 11175364; Prasad D D, Rao V N, Reddy ES. Structure and expression of human Fli-1 gene. Cancer Res. 1992;52(20):5833-7). In addition, an approach to targeting FLI1 is to disruptit from protein interactions with YK-4-279 rather than eliminating itsexpression.

YK-4-279 Inhibits the Function of Ets Family Members ERG, ETV1, andEWS-FLI1

In the childhood/young adult cancer, Ewing sarcoma, the EWStranscription activation domain is fused to an ets family member leadingto the novel fusion protein, EWS-FLI1. We identified and validated smallmolecule YK-4-279 that prevents the binding of EWS-FLI1 to RHA leadingto cellular apoptosis in a panel of Ewing sarcoma cell lines (Erkizan HV, Kong Y, Merchant M, Schlottmann S, Barber-Rotenberg J S, Yuan L, etal. A small molecule blocking oncogenic protein EWS-FLI1 interactionwith RNA helicase A inhibits growth of Ewing's sarcoma. Nat Med. 2009July; 15(7):750-6. PubMed PMID: 19584866. eng). We also demonstratedreduced tumor growth in Ewing Sarcoma xenograft models while notaffecting the growth of non-EWS-FLI1 containing tumors at similardosages. As an important proof of specificity, only the (S) enantiomerof YK-4-279 is able to inhibit the functional activities of EWS-FLI1including binding to RHA, transcript activation, and alternativesplicing (Barber-Rotenberg J S, Selvanathan S P, Kong Y, Erkizan H V,Snyder T M, Hong P S, et al. Single Enantiomer of YK-4-279 DemonstratesSpecificity in Targeting the Oncogene EWS-FLI1. Oncotarget. 2012February; 3(2):172-82. PubMed PMID: 22383402. Epub 2012/03/03. eng).Advanced prostate cancers over-express ERG, ETV1 or ETV4 by eitherchromosomal translocation or gene amplification. ERG, ETV1, or ETV4activity have been directly implicated to increase invasion andmetastasis. All three are ets family proteins that share significanthomology to FLI1, and have essentially identical DNA binding domains.Prostate cells driven by ERG or ETV1 showed significantly decreasedinvasion when treated with YK-4-279 (Rahim S, Beauchamp E M, Kong Y,Brown M L, Toretsky J A, Uren A. YK-4-279 Inhibits ERG and ETV1 MediatedProstate Cancer Cell Invasion. PLoS ONE. 2011; 6(4):e19343. PubMed PMID:21559405. Pubmed Central PMCID: 3084826. Epub 2011/05/12. eng). Thiscross-tumoral activity based upon the homology of ets transcriptionfactors led us to explore additional tumors that might be in part drivenby an ets transcription factor.

The ets family member FLI1 may therefore be a novel molecular target andYK-4-279 a potential targeted therapeutic in GBM. Orthotopic xenograftand genetically engineered mouse models of GBM are helpful forproof-of-principle studies to support a rationale for advancement tohuman clinical trials. FLI1 may be a novel vital target for GBM and thatYK-4-279 may be useful as a future therapeutic.

GBM is one of the targeted tumor types of The Cancer Genome Atlas(TCGA), with multiple dataset available for analysis. Alterations inFLI1 as well as highly homologous proteins have been searched and it wasfound that 23% of GBM specimens had alterations that support FLI1 as anovel target (FIG. 8).

Considering the close homology between FLI1, ERG and ETV1, the bindingof YK-4-279 to ERG and ETV1 was evaluated (Rahim S, Beauchamp E M, KongY, Brown M L, Toretsky J A, Uren A. YK-4-279 Inhibits ERG and ETV1Mediated Prostate Cancer Cell Invasion. PLoS ONE. 2011; 6(4):e19343.PubMed PMID: 21559405. Pubmed Central PMCID: 3084826. Epub 2011/05/12.eng). The affinity (KD) of YK-4-279 for EWS-FLI1 was measured to be 9.5μM (Erkizan H V, Kong Y, Merchant M, Schlottmann S, Barber-Rotenberg JS, Yuan L, et al. A small molecule blocking oncogenic protein EWS-FLI1interaction with RNA helicase A inhibits growth of Ewing's sarcoma. NatMed. 2009 July; 15(7):750-6. PubMed PMID: 19584866. Eng;Barber-Rotenberg J S, Selvanath). The steady state kinetics of YK-4-279binding to recombinant ERG and ETV1 using surface plasmon resonance hada binding affinity (KD) of 11.7 μM for ERG and 17.4 μM for ETV1, whereasit bound the non-specific protein BSA with a weak affinity of 122.4 μM.FIG. 9 shows that YK-4-279 binds to ERG and ETV1 with a KD of 11.7 μMand 17.9 μM respectively. Steady state kinetics were measured on aBiacore T100 instrument, as previously described (Erkizan H V, Kong Y,Merchant M, Schlottmann S, Barber-Rotenberg J S, Yuan L, et al. A smallmolecule blocking oncogenic protein EWS-FLI1 interaction with RNAhelicase A inhibits growth of Ewing's sarcoma. Nat Med. 2009 July;15(7):750-6. PubMed PMID: 19584866. Eng; Barber-Rotenberg J S,Selvanath), SPR sensograms are not shown.

A large sequencing project of GBM that included analysis of XX celllines has been completed (Solomon D A, Kim T, Diaz-Martinez L A, Fair J,Elkahloun A G, Harris B T, et al. Mutational inactivation of STAG2causes aneuploidy in human cancer. Science. 2011 Aug. 19;333(6045):1039-43. PubMed PMID: 21852505. Epub 2011/08/20. eng). Sevencell lines were selected with a spectrum of genetic abnormalities thatoccur in GBM. Table 3 shows the heterogeneity of GBM cell lines. A panelof GBM cell lines were acquired that represent the heterogeneity of thedisease. The (+) indicates the expression of the listed protein, eitherwild-type or mutant. The (−) indicates the absence of expression on animmunoblot. These cell lines were used to evaluate the expression ofFLI1 as well as sensitivity to the inhibitor YK-4-279 (FIG. 3).

TABLE 3 DKMG DBTRG 42MGBA GAMG U87MG H4 8MGBA EGFR + − + + + − + Myc +− + + + + + PTEN + − − + + − + MDM2 + + − + − − − p53 + + + + − − +p14ARF − − − − − − + 21WAF1/CIP + + + + + + + CDK4 + + + + + + +CDK6 + + + + − + + p16INK4a − − − − − − + p18INK4c + + + + − + + RB + +− + + + −

Six of the 7 GBM cell lines (85%) demonstrated FLI1 expression byimmunoblot (FIG. 10, top panel). Growth of each of these cell lines wasreduced by YK-4-279 with IC50 ranging from 0.5 up to 9.9 μM and aninverse correlation was observed between the level of FLI1 and thesensitivity to YK-4-279 (r2=0.8, FIG. 10).

To evaluate the potential of FLI1 as a GBM target, two transgenic modelswere analyzed (Chow L M, Endersby R, Zhu X, Rankin S, Qu C, Zhang J, etal. Cooperativity within and among Pten, p53, and Rb pathways induceshigh-grade astrocytoma in adult brain. Cancer Cell. 2011 Mar. 8;19(3):305-16. PubMed PMID: 21397855. Pubmed Central PMCID: 3060664).Very low expression for two FLI1 probe sets comparing normal brainstem,brainstem astrocytes, and cortical astrocytes was observed (FIG. 11).However, 20 of 22 double knock-out (PTEN/p53) and 13 of 14 tripleknockout (PTEN/p53/Rb) tumors had significant expression of the FLI1based upon the two probesets analyzed (FIG. 11).

In order to determine the FLI1 expression level in human GBM, a panel ofGBM was stained with FLI1 antibody. The panel consisted of six randomlyselected grade 4 tumors; four of six (66%) showed convincing IHCstaining for FLI1 after an antibody was optimized to eliminatecross-reacting proteins and non-specific signal (FIG. 12). Fouradditional tumors were considered unevaluable since the internalpositive control, endothelial cells, were not positive.

One of the significant challenges in getting novel targeted therapy intoGBM is overcoming the blood-brain barrier (BBB). As part of apharmacokinetic evaluation of YK-4-279, tissue levels were measured andcompared these with plasma in 12 mice that received 75 mg/kg IV racemicYK-4-279. Levels of YK-4-279 in brain tissue were 74% that of the Ewingsarcoma pretibial xenograft tumor, which would be adequate forinhibiting FLI1. In addition, when rat pharmacokinetics were performedusing IV injection of compound, rats became somnolent after rapidinjection, which did not occur with slower infusion, thus supporting anability to pass into the central nervous system across the BBB.

The data provided herein identifies FLI1 as a putative target for GBM.The combination of TCGA data, a panel of cell lines, GEMM model, andpanel of IHC from human tumors support further validation of FLU.

Validation of FLI1 as a Novel Target in GBM

FLI1 as putative target is validated by evaluating whether it isnecessary for GBM cell growth. Whether FLI1 is a potential oncogene inglial stem cells is determined. GBM cells are compared with normal humanastrocytes and glial stem cells for the importance of FLI1 in order toaddress the therapeutic index of targeting FLU. The comparison withnormal brain cells is useful to establish FLI1 as a valid target with apreferable therapeutic index.

GBM Cell Lines Require FLI1 Expression for Survival, Growth, andInvasion are Identified

Using an shRNA vector that is tagged with EGFP which targets the 3′UTRof FLI1 GBM cell lines require FLI1 expression for survival, growth, andinvasion are identified. The shRNA are infected into cells using alentiviral system. Thus, the relative importance of FLI1 in seven GBMcell lines which span much of the genotypic heterogeneity is evaluated(Table 3). After FLI1 is reduced with shRNA, changes are measure betweenscrambled shRNA control and FLI1 reduction in monolayer growth,invasion, anchorage-independent growth, and tumorigenesis assays. Cellculture experiments are performed as previously reported (Erkizan H V,Kong Y, Merchant M, Schlottmann S, Barber-Rotenberg J S, Yuan L, et al.A small molecule blocking oncogenic protein EWS-FLI1 interaction withRNA helicase A inhibits growth of Ewing's sarcoma. Nat Med. 2009 July;15(7):750-6. PubMed PMID: 19584866. Eng; Rahim S, Beauchamp E M, Kong Y,Brown M L, Toretsky J A, Uren A. YK-4-279 Inhibits ERG and ETV1 MediatedProstate Cancer Cell Invasion. PLoS ONE. 2011; 6(4):e19343. PubMed PMID:21559405. Pubmed Central PMCID: 3084826. Epub 2011/05/12. eng). Invasionassays are performed using tumor cells and their invasion throughumbilical endothelial cells using an electrical-impedance basedtechnique that monitors and quantifies in real-time the invasion ofendothelial cells by malignant tumor cells. The xCELLigence instrument,manufactured by Roche, which measures changes in electrical impedance ascells attach and then as tumor cells disrupt this attachment is used(Rahim S, Beauchamp E M, Kong Y, Brown M L, Toretsky J A, Uren A.YK-4-279 Inhibits ERG and ETV1 Mediated Prostate Cancer Cell Invasion.PLoS ONE. 2011; 6(4):e19343. PubMed PMID: 21559405. Pubmed CentralPMCID: 3084826. Epub 2011/05/12. Eng; Rahim S, Uren A. A real-timeelectrical impedance based technique to measure invasion of endothelialcell monolayer by cancer cells. Journal of visualized experiments: JoVE.2011 (50). PubMed PMID: 21490581. Pubmed Central PMCID: 3169283).Xenograft experiments use polyclonal shRNA reduced FLI1 in all sevencell lines. Each shRNA FLI1 and scrambled cell lines arestereotactically injected into 5 athymic mice (assisted by Fiandanca).(7 cell lines, 5 animals per cell line, +/−FLI1=70). Growth is monitoredby MRI in the GU Animal Imaging shared resource at 7-10 day intervals.Calculations of tumor growth kinetics are performed by region ofinterest analysis as described, using Bruker Paravision 5.0 software orImageJ (NIH) (Truong A H, Cervi D, Lee J, Ben-David Y. Directtranscriptional regulation of MDM2 by Fli-1. Oncogene. 2005 Feb. 3;24(6):962-9. PubMed PMID: 15592502; Pimanda J E, Chan W Y, Donaldson U,Bowen M, Green A R, Gottgens B. Endoglin expression in the endotheliumis regulated by Fli-1, Erg, and Elf-1 acting on the promoter and a −8-kbenhancer. Blood. 2006 Jun. 15; 107(12):4737-45. PubMed PMID: 16484587).Growth statistics of tumors are calculated to build a time-dependentprofile of progression and treatment.

Transfection of Normal Human Astrocytes with a Full-Length FLI1 cDNA

To determine the transformative effect of FLI1 upon astrocytes, normalhuman astrocytes are transfected with a full-length FLI1 cDNA usinglentiviral system and evaluate for transformation in soft agar and invivo orthotopic injection assays. Control (empty vector) and FLI1transfected polycolonal cells are placed in soft agar foranchorage-independent growth assays (Erkizan H V, Kong Y, Merchant M,Schlottmann S, Barber-Rotenberg J S, Yuan L, et al. A small moleculeblocking oncogenic protein EWS-FLI1 interaction with RNA helicase Ainhibits growth of Ewing's sarcoma. Nat Med. 2009 July; 15(7):750-6.PubMed PMID: 19584866. eng). Xenograft studies are performed asdescribed above. (2 cell lines+/−FLI1, five animals per cell line=20animals). Animals are imaged by MRI to assess tumor growth, as describedabove, every 10 days.

Glial stem cells are evaluated for both for their innate expression ofFLI1 and the evaluation of oncogenic effects when FLI1 is exogenouslyexpressed. Published (Lelievre E, Lionneton F, Mattot V, Spruyt N,Soncin F. Ets-1 regulates fli-1 expression in endothelial cells.Identification of ETS binding sites in the fli-1 gene promoter. J BiolChem. 2002 Jul. 12; 277(28):25143-51. PubMed PMID: 11991951) and novelcell lines are used. To evaluate anchorage-independent growth andinvasion assays, for both control and FLU expression are performed. Inaddition, these cell lines are used in xenograft experiments, bothtransfected with control and FLI1 expression (always proven byimmunoblots prior to evaluation). These cells are all be carefully grownin minimal growth media with exogenous growth factors rather than serumto maintain their pristine neural qualities (Rossi S, Orvieto E,Furlanetto A, Laurino L, Ninfo V, Dei Tos A P. Utility of theimmunohistochemical detection of FLI-1 expression in round cell andvascular neoplasm using a monoclonal antibody. Mod Pathol. 2004 May;17(5):547-52. PubMed PMID: 15001993). (3 cell lines+/−FLI1, five animalsper cell line=15 animals).

The correlation between YK-4-279 toxicity and FLI1 levels is measured.YK-4-279 targets the FLI1 component of EWS-FLI1 (Barber-Rotenberg J S,Selvanathan S P, Kong Y, Erkizan H V, Snyder T M, Hong P S, et al.Single Enantiomer of YK-4-279 Demonstrates Specificity in Targeting theOncogene EWS-FLI1. Oncotarget. 2012 February; 3(2):172-82. PubMed PMID:22383402. Epub 2012 Mar. 3. Eng; Rahim S, Beauchamp E M, Kong Y, Brown ML, Toretsky J A, Uren A. YK-4-279 Inhibits ERG and ETV1 MediatedProstate Cancer Cell Invasion. PLoS ONE. 2011; 6(4):e19343. PubMed PMID:21559405. Pubmed Central PMCID: 3084826. Epub 2011 May 12. eng).Following an evaluation of the effect of FLI1 inhibitor YK-4-279 upon apanel of cell lines and correlation of inhibition with FLI1 expression,these results are compared to the shRNA data. Preliminary data for 7 GBMcell lines with correlation to FLI1 expression. This is expanded to morecell lines with methodology used to generate FIG. 9. Non-tumor glialcell lines are included for these comparisons.

Whether FLI1 expression correlates with other known GBM genotypes andphenotypes is determined. A series of on-line informatics tools fromTCGA as well as correlation with our cell line data is used. Theanalysis includes FLI1 cDNA or protein expression and evaluates whetherFLI1 correlates with other known GBM genetic events, such as loss ofPTEN, p16, p53 mutation, IDH1 point mutation or EGFR mutation. This isexpanded to include patient tumors that are resected at diagnosis andinclusion of FLI1 into the panel of genetic markers evaluated.

Utilizing Animal Models of GBM to Evaluate YK-4-279 as a Therapy

To establish whether YK-4-279 is effective in either slowing growth orcausing regression of GBM, YK-4-279 is administered to mice withestablished GBM. This evaluates both xenograft and transgenic models ofGBM.

To establish a method for evaluating intracranial lesions in groups ofmice, rather than one at a time with MM. While the MRI provides a highlydetailed graphic and metabolic spectrum of GBM tumors, the time toscreen mice limits its use for larger studies. Two GBM cell lines arecreated, selected from the GBM orthotopic screening of seven cell linesthat are used for Xenogen intracranial imaging. The two GBM cell linesare selected based upon their requirements for FLI1, as determined aboveand growth kinetics. These two GBM cell lines are stably transfectedwith luciferase so that groups of animals can be screened/monitoredusing the Xenogen system. For more detailed studies and volumetriccomparison, selected animals are evaluated using MM. Cell lines arescreened in vitro followed by an orthotopic xenograft pilot study (2cell lines×5 animals=10 animals). Prior to Xenogen imaging, animals areinjected with luciferase substrate intraperitoneally.

YK-4-279 upon xenograft GBM tumors is evaluated by treating animalsseven days after tumor injection and those with symptomatic tumors.Early tumors are screened with Xenogen, and size correlated with MRI,and treatment started when animals 5 mm³ lesions. YK-4-279 passes theblood-brain barrier. Animals are treated with BID injections of YK-4-279similar to the dose that led to regression of Ewing sarcoma tumors (FIG.13). The study will measure brain tumor volumes, animal symptoms, andoverall survival. At necropsy, tumors and normal adjacent brain will beevaluated by immunohistochemistry for GBM markers, apoptosis, and FLI1regulated target genes. FIG. 13 illustrates that three days of treatmentwith (S)-YK-4-279 or racemic shows significant tumor regression. FIG.13A: Mice with ES xenografts were treated with 400 mg/kg compound orcontrols as indicated. Starting well-established tumors (300 mm³), micewere treated with intraperitoneal compound for three days, 6 totaldoses. FIG. 13B: H and E stained tumors from same experiment.

Since the vast majority of patients with GBM present with symptoms andrelatively large tumors, YK-4-279 is tested against larger, symptomatictumors. Thus, YK-4-279 is evaluated on upon well-established GBM, 20mm³, by MM, by treating animals after tumors are established and atfirst signs of symptoms. Tumors at this time are detectable withXenogen. Animals undergo Xenogen evaluation twice a week while ontreatment. The study compares tumor volumes, animal symptoms, andoverall survival. At necropsy, tumors and normal adjacent brain areevaluated by immunohistochemistry for GBM markers, apoptosis, and FLI1regulated target genes.

The GEMM model of GBM is used. Animals are bred as published (Chow L M,Endersby R, Zhu X, Rankin S, Qu C, Zhang J, et al. Cooperativity withinand among Pten, p53, and Rb pathways induces high-grade astrocytoma inadult brain. Cancer Cell. 2011 Mar. 8; 19(3):305-16. PubMed PMID:21397855. Pubmed Central PMCID: 3060664). At approximately 90 days oflife, animals have MRI evaluation every 10-14 days to look for onset ofGBM. In Experiment 1 animals are treated with YK-4-279 or controlstarting at day 90 (10 animals in control and treated=20 animals). InExperiment 2 animals are treated at the onset of symptoms or MRImeasured tumor of greater than 2 mm in any dimension (10 animals incontrol and treated=20 animals). Following treatment, animals areevaluated as described above. Administration of YK-4-279 is using theintraperitoneal route.

Data is provided to support further exploration of FLI1 and potentiallyother ets family members as drivers of GBM.

References pertaining to selected cancers include the following: CBTRUSStatistical Report: Primary Brain and Central Nervous System TumorsDiagnosed in the United States in 2004-2008 (Mar. 23, 2012 Revision).Central Brain Tumor Registry of the United States [Internet]. 2012;http://www.cbtrus.org. Available from: http://www.cbtrus.org.

Example 11—Use of YK-4-279 for Treating Lung Cancer

Epithelial-to-mesenchymal transition (EMT) is a key component of thepathogenesis of carcinomas. EMT induces significant changes in cellmorphology and behavior that impart metastatic and drug-resistantphenotypes. Moreover, there is evidence suggesting that EMT participatesin the generation of cancer stem cells. Lung cancer is the leading causeof cancer-related mortality, mainly because it is typically diagnosed atadvanced stages that are difficult to treat. Advances in ourunderstanding of the molecular genetics of cancers have identifiedindividual molecules required for tumorigenesis. This has led to thedevelopment of targeted therapies that are successful for treatingcertain cancers. Examples of these molecular targets include cellsurface growth factor receptors and intracellular protein tyrosinekinases. Unfortunately, such treatments have not significantly improvedoverall survival or quality of life for patients with lung cancer.

Recent discoveries described here have led to the hypothesis that theproduct of the E-26 Transforming Sequence (ETS)-related gene ERG, amember of the ETS family of transcription factors, plays an importantrole in EMT. Moreover, it induces EMT and the malignant progression ofepithelial cells through direct up-regulation of the expression of zincfinger E-box binding homeobox 1 and 2 genes (ZEB1, ZEB2). ZEB1 is linkedto EMT in lung cancer cells, and inhibiting its expression using siRNAnot only reverses EMT but also inhibits tumor growth in vitro and invivo. Because lung cancer cells express high levels of ERG, ERG mayinduce EMT through ZEB. Experiments are conducted that determine whetherERG participates in EMT of lung cancer cells mediated by ZEB1/2. Newformulations of YK-4-279 are produced and evaluated for treating lungcancer.

ERG as a transcription factor modulates expression of many genes thatare important for carcinogenesis. Earlier observations suggested thatoncogenic properties of ERG include its ability to induceepithelial-to-mesenchymal transition (EMT). In different experimentalsystems, ERG has been shown to induce expression of zinc finger E-boxbinding homeobox 1 and 2 genes (ZEB1, ZEB2), which are positiveregulators of EMT in cancer cells. Since EMT results in metastasis anddrug resistance in NSCLC, inhibition of molecular pathways leading toEMT may have significant clinical utility.

The role of ERG in mediating EMT in lung cancer cells is determined. EMTand drug resistance phenotypes of NSCLC cell lines in response tochanges in ERG expression is determined. If ERG induces EMT like it doesin other epithelial tumors is determined. Further, if the ERG mediatedEMT in NSCLC is through ZEB1/2 genes is determined. These experimentsinvolve inhibition of ERG and ZEB expression in NSCLC cells by RNAitechnologies. EMT phenotype is evaluated by real-time PCR and westernblotting for established EMT markers.

Formulations of YK-4-279 that can be administered parenterally areproduced and the effects of YK-4-279 on the proliferation and malignantproperties of lung cancer cells are determined. An examples excipient isβ-hydroxypropyl cyclodextrin ((3-HPCD). NSCLC cells are treated withYK-4-279 and their response is measured in multiple in vitro and in vivomodels. Cell viability, chemotaxis, endothelial cell invasion andxenograft growth in immunocompromised mice is measured. The potentialsynergy between YK-4-279 and most common chemotherapeutic agents forNSCLC is determined. The properties of drug resistance and highmetastatic potential of NSCLC cells, mediated by EMT, contributesignificantly to the poor prognosis of patients with NSCLC.

The properties of drug resistance and high metastatic potential of NSCLCcells, mediated by EMT, contribute significantly to the poor prognosisof patients with NSCLC. A specific protein to reverse the EMT phenotypein lung cancer is targeted using a small molecule. FIG. 14 illustratesERG induces expression of ZEB1 and ZEB2, which activate EMT leading tolung cancer metastasis and drug resistance.

Targeting drugs to specific molecules required for the growth of cancercells remains a difficult challenge despite recent advances in molecularand cellular biology. Although proteins that drive the unregulatedreproduction of cancer cells are known, only a few have served astargets of effective therapies. Examples include an intracellularprotein tyrosine kinase whose activity is inhibited by a small moleculecalled imatinib mesylate used to treat chronic myelogenous leukemia; anda monoclonal antibody (trastuzumab) used to treat breast cancers, istargeted to a cell surface growth factor receptor. These limited butsignificant and highly encouraging successes have stimulated continuingand robust research by the pharmaceutical industry. Inhibiting theactivity of an enzyme or the activation of receptor are well-establishedgoals of drug development for numerous diseases in addition to cancer,because the biochemistry of these proteins is so well understood. Incontrast, the biochemistry involved in the binding of proteins to oneanother is much more complex and poorly understood, and inhibiting theseinteractions has therefore received relatively little attention.

ERG is a further challenge for designing a targeted therapy, because itlocalizes to the nucleus and lacks enzyme activity. YK-4-279 is a smallmolecule that inhibits ERG's transcriptional activity, and isinvestigated for its role in NSCLC. Whether YK-4-279, by binding to andinterfering with ERG function required for EMT is determined (FIG. 14),can be used to treat NSCLC.

ERG is an oncogenic protein. The E-26 Transforming Sequence(ETS)-related gene ERG encodes a member of the ETS family oftranscription factors that is essential for endothelial homeostasis,differentiation, and angiogenesis in many tissues (Liu F, Patient R.Genome-wide analysis of the zebrafish ETS family identifies three genesrequired for hemangioblast differentiation or angiogenesis. Circulationresearch. 2008; 103:1147-54; Sashida G, Bazzoli E, Menendez S, Liu Y,Nimer S D. The oncogenic role of the ETS transcription factors MEF andERG. Cell cycle. 2010; 9:3457-9). Evidence suggests that the activitiesof certain genes that are regulated by ERG are required forangiogenesis. For example, VE-cadherin, which requires ERG for itsexpression, is essential for endothelial junctional stability andendothelial survival, both critical processes in angiogenesis (Yuan L,Sacharidou A, Stratman A N, Le Bras A, Zwiers P J, Spokes K, et al. RhoJis an endothelial cell-restricted Rho GTPase that mediates vascularmorphogenesis and is regulated by the transcription factor ERG. Blood.2011; 118:1145-53). In carcinogenesis, ETS transcription factors areinvolved in the regulation of numerous genes that participate inprocesses required for metastasis, including degradation of theextracellular matrix, and formation of cell-to-cell and cell-to-matrixjunctions (Lelievre E, Lionneton F, Soncin F, Vandenbunder B. The Etsfamily contains transcriptional activators and repressors involved inangiogenesis. The international journal of biochemistry & cell biology.2001; 33:391-407).

Specific examples include the receptor for vascular endothelial growthfactor, endoglin, matrix metalloproteinases, collagenase 1, and hemeoxygenase 1. ERG is overexpressed in hematopoietic and epithelial cellcancers and acts as a potent oncogene in human prostate cancers (Chen Y,Chi P, Rockowitz S, Iaquinta P J, Shamu T, Shukla S, et al. ETS factorsreprogram the androgen receptor cistrome and prime prostatetumorigenesis in response to PTEN loss. Nature medicine. 2013; Rahim S,Uren A. Emergence of ETS transcription factors as diagnostic tools andtherapeutic targets in prostate cancer. American journal oftranslational research. 2013; 5:254-68; Turner D P, Watson D K. ETStranscription factors: oncogenes and tumor suppressor genes astherapeutic targets for prostate cancer. Expert review of anticancertherapy. 2008; 8:33-42).

ERG and EMT signifies poor clinical outcome in NSCLC. Notable findingsleading to our interest in the role of ERG in lung cancers include thedetection of relatively high levels of ERG expression in NSCLCs and thepresence of alternatively spliced versions of ERG in 100% of lung tumorsamples compared with normal tissue (Xi L, Feber A, Gupta V, Wu M,Bergemann A D, Landreneau R J, et al. Whole genome exon arrays identifydifferential expression of alternatively spliced, cancer-related genesin lung cancer. Nucleic acids research. 2008; 36:6535-47). Analysis ofmRNA expression by micro array: ERG mRNA expression ranking is in top 8%(Ramaswamy S, Ross K N, Lander E S, Golub T R. A molecular signature ofmetastasis in primary solid tumors. Nature genetics. 2003; 33:49-54),and in top 11% (Ding L, Getz G, Wheeler D A, Mardis E R, McLellan M D,Cibulskis K, et al. Somatic mutations affect key pathways in lungadenocarcinoma. Nature. 2008; 455:1069-75) in NSCLC tissue samples.Since ERG target genes are involved in EMT phenotype we hypothesizedthat ERG mediated EMT may contribute to malignant phenotype of NSCLC.

EMT was first described in early embryonic development when cells losetheir epithelial characteristics and acquire mesenchymal phenotypes(Sato M, Shames D S, Hasegawa Y. Emerging evidence ofepithelial-to-mesenchymal transition in lung carcinogenesis.Respirology. 2012; 17:1048-59). As EMT progresses, cells acquire a moremotile and invasive phenotype. Therefore, EMT emerged as an importantcomponent of carcinogenesis. The associations between EMT and NSCLClocal invasion, angiogenesis, distant metastasis as well as drugresistance and anti-apoptotic phenotypes have been demonstrated bynumerous studies conducted in vivo and in vitro (Table 4). For example,the expression of molecules involved in EMT correlate with theclinico-pathological features of NSCLC, including increased metastasisand shortened overall survival of patients (Dauphin M, Barbe C, LemaireS, Nawrocki-Raby B, Lagonotte E, Delepine G, et al. Vimentin expressionpredicts the occurrence of metastases in non small cell lung carcinomas.Lung cancer. 2013; 81:117-22). Moreover, the important role of EMT incarcinogenesis is indicated by cellular phenotypes characteristic ofstem cells (Mani S A, Guo W, Liao M J, Eaton E N, Ayyanan A, Zhou A Y,et al. The epithelial-mesenchymal transition generates cells withproperties of stem cells. Cell. 2008; 133:704-15). Taken together, thesestudies provide strong justification for inhibiting EMT that occurs inthe development of lung cancer. Table 4 lists molecules involved in EMTthat correlate with clinical features in NSCLC.

TABLE 4 EMT Genes Clinical features References Epithelial Longer overallNakata S, Sugio K, Uramoto H, Oyama T, Cadherin survival Hanagiri T,Morita M, et al. The methylation status and protein expression of CDH1,p16(INK4A), and fragile histidine triad in nonsmall cell lung carcinoma:epigenetic silencing, clinical features, and prognostic significance.Cancer. 2006; 106: 2190-9. Negative for lymph Kase S, Sugio K, YamazakiK, Okamoto T, node metastasis Yano T, Sugimachi K. Expression of E-cadherin and beta-catenin in human non-small cell lung cancer and theclinical significance. Clinical cancer research: an official journal ofthe American Association for Cancer Research. 2000; 6: 4789-96. SLUGPostoperative relapse Shih JY, Tsai MF, Chang TH, Chang YL, Yuan A, YuCJ, et al. Transcription repressor slug promotes carcinoma invasion andpredicts outcome of patients with lung adenocarcinoma. Clinical cancerresearch: an official journal of the American Association for CancerResearch. 2005; 11: 8070-8. Shorter overall Chiou SH, Wang ML, Chou YT,Chen CJ, survival Hong CF, Hsieh WJ, et al. Coexpression of Oct4 andNanog enhances malignancy in lung adenocarcinoma by inducing cancer stemcell- like properties and epithelial-mesenchymal transdifferentiation.Cancer research. 2010; 70: 10433-44. SNAIL Shorter overall Yanagawa J,Walser TC, Zhu LX, Hong L, survival Fishbein MC, Mah V, et al. Snailpromotes CXCR2 ligand-dependent tumor progression in non-small cell lungcarcinoma. Clinical cancer research: an official journal of the AmericanAssociation for Cancer Research. 2009; 15: 6820-9. TWIST Shorter overallHung JJ, Yang MH, Hsu HS, Hsu WH, Liu JS, survival Wu KJ. Prognosticsignificance of hypoxia- HIF-1 alpha Shorter overall induciblefactor-1alpha, TWIST1 and Snail survival expression in resectablenon-small cell lung Shorter recurrence cancer. Thorax. 2009; 64: 1082-9.free survival

ERG target gene ZEB1 mediates EMT. EMT is a complex cellular responsethat involves multiple signaling pathways. The zinc finger E-box-bindinghomeobox (ZEB) proteins are key regulators of EMT (Takeyama Y, Sato M,Horio M, Hase T, Yoshida K, Yokoyama T, et al. Knockdown of ZEB1, amaster epithelial-to-mesenchymal transition (EMT) gene, suppressesanchorage-independent cell growth of lung cancer cells. Cancer letters.2010; 296:216-24). In particular, ZEB1 plays a predominant role in theEMT-associated carcinogenic phenotypes of lung cancer by regulating theexpression of genes that encode proteins that participate in EMT. Forexample, inhibition of ZEB1 expression by siRNA in lung cancer celllines results in the reversal of EMT, increased sensitivity todocetaxel, and reduced growth of lung cancer cells in vitro and in vivo(Ren J, Chen Y, Song H, Chen L, Wang R. Inhibition of ZEB1 reverses EMTand chemoresistance in docetaxel-resistant human lung adenocarcinomacell line. Journal of cellular biochemistry. 2013; 114:1395-403). Mostimportant, ERG mediates EMT in prostate cancer cells through the ZEBaxis (Leshem O, Madar S, Kogan-Sakin I, Kamer I, Goldstein I, Brosh R,et al. TMPRSS2/ERG promotes epithelial to mesenchymal transition throughthe ZEB1/ZEB2 axis in a prostate cancer model. PloS one. 2011;6:e21650). Moreover, miR-30 suppresses EMT in prostate cancer cells bydirectly targeting ERG expression (Kao C J, Martiniez A, Shi X B, YangJ, Evans C P, Dobi A, et al. miR-30 as a tumor suppressor connectsEGF/Src signal to ERG and EMT. Oncogene. 2013). These and other studiesindicate that it is reasonable to conclude that efforts to target ZEB1and ZEB2 to reverse EMT in lung cancer may be successful. Because RNAitechnologies are not advanced enough for clinical applications, it willbe necessary to identify alternative mechanisms to inhibit ZEB1expression in lung cancers. It is known that ERG binding sites arepresent in the ZEB1 and ZEB2 promoter regions (Leshem O, Madar S,Kogan-Sakin I, Kamer I, Goldstein I, Brosh R, et al. TMPRSS2/ERGpromotes epithelial to mesenchymal transition through the ZEB1/ZEB2 axisin a prostate cancer model. PloS one. 2011; 6:e21650). In 2009 wediscovered YK-4-279 as an inhibitor of EWS-FLI1, a fusion proteinencoded by a tumor-specific rearranged gene in Ewing Sarcoma (Erkizan HV, Kong Y, Merchant M, Schlottmann S, Barber-Rotenberg J S, Yuan L, etal. A small molecule blocking oncogenic protein EWS-FLI1 interactionwith RNA helicase A inhibits growth of Ewing's sarcoma. Nature medicine.2009; 15:750-6). More recently, based on the homology between two ETSmembers, FLI1 and ERG, YK-4-279 binds directly to ERG and inhibits itstranscriptional activity (Rahim S, Beauchamp E M, Kong Y, Brown M L,Toretsky J A, Uren A. YK-4-279 inhibits ERG and ETV1 mediated prostatecancer cell invasion. PloS one. 2011; 6:e19343). Inhibition of ERGfunction in lung cancer cells may reverse EMT mediated by ZEB proteinsand lead to inhibition of metastatic growth and increased sensitivity tochemotherapeutic drugs.

YK-4-279 Binds ERG

YK-4-279, a small molecule that binds directly to EWS-FLI1 and inhibitsthe growth of Ewing Sarcoma cells (Erkizan H V, Kong Y, Merchant M,Schlottmann S, Barber-Rotenberg J S, Yuan L, et al. A small moleculeblocking oncogenic protein EWS-FLI1 interaction with RNA helicase Ainhibits growth of Ewing's sarcoma. Nature medicine. 2009; 15:750-6).EWS-FLI1 is the product of a chromosomal translocation. FLI1 is an ETSfamily transcription factor with a conserved DNA binding domain.Alignment of FLI1 with another ETS family member, ERG, amino acidsequence shows significant similarities (63.5% identity, 80.2%homology). Commercially available recombinant ERG protein (Origene,Rockville, Md.) was obtained and measured direct binding affinity forYK-4-279 on a Biacore T-100 (FIG. 15), which detects molecularinteractions in real time without a reporter moiety. Recombinant proteinwas immobilized on Biacore microchips and binding was measured in thepresence of varying YK-4-279 concentrations. We detected YK-4-279binding to ERG with a steady state KD of 11.7 μM. FIG. 15 illustratesYK-4-279 directly interacts with ERG protein. Purified recombinant ERGwas immobilized on Biacore CMS microchips, and direct binding to eightdifferent YK-4-279 concentrations (0.1-50 μM) was determined by SPR.Steady state KD was calculated using Biaevaluation software.

YK-4279 inhibits transcriptional activity of ERG. ETS proteins controlexpression of target genes encoding proteins that participate in diversebiochemical processes, many of which contribute to oncogenic growth(Hollenhorst P C, McIntosh L P, Graves B J. Genomic and biochemicalinsights into the specificity of ETS transcription factors. Annualreview of biochemistry. 2011; 80:437-71). The effect of YK-4-279 onERG's transcriptional activity was tested utilizing promoter reporterassays and endogenous gene expression profiling. YK-4-279 inhibited ERGactivation of an ETS target gene promoter (Id2) controlling luciferaseexpression in COST cells (FIG. 16A). VCaP prostate cancer cells possessthe TMPRSS2/ERG fusion gene, where ERG induces expression of specificendogenous target genes such as PLAU, ADAM19 and PLAT. Real time(RT)-PCR analysis revealed that YK-4-279 significantly inhibited theirexpression but not that of ERG expression (FIG. 16B). These findingswere extended to the protein level for ERG and PLAU. Inhibition byYK-4-279 was comparable to that observed when cells were treated withERG siRNA (FIG. 16B) (Rahim S, Beauchamp E M, Kong Y, Brown M L,Toretsky J A, Uren A. YK-4-279 inhibits ERG and ETV1 mediated prostatecancer cell invasion. PloS one. 2011; 6:e19343). Since epithelial cellsdo not express FLI1, YK-4-279's effects were most likely due toinhibiting ERG. This provides further evidence that YK-4-279 is notsimply a general inhibitor of transcription and translation (ERG mRNAand protein levels did not change), but it specifically inhibits thetranscriptional activities of ETS family proteins.

ERG is Expressed in NSCLC Cell Lines and Induces EMT Markers

Five NSCLC cell lines were examined to confirm that significant ERGexpression is present similar to what was observed in human tumorsamples. A western blot analysis of A549, H1944, H358, H1395, and H596cell lysates were performed (FIG. 17). Four out of 5 cell linesexpressed high levels of ERG protein. H358 cell line that expressed verylittle ERG will be used as a negative control in our studies.

Earlier work in other tumor types suggested that ERG may induce EMT. Inorder to test, if the same effect exists in NSCLC cells, an ERGexpression vector was transfected to H358 cells, which has relativelyvery low levels of endogenous ERG protein (FIG. 18A). When the H358cells expressed high level of ERG protein, we observed a significantincrease in expression of two EMT markers, ZEB1 and Foxc2 (FIG. 18B),suggesting that ERG can induce EMT in NSCLC cells.

FIGS. 18A and 18B illustrate that ERG expression induces EMT markers.H358 NSCLC cells were transfected with a cDNA coding for human ERGprotein. Increased ERG expression was detected by western blotting (FIG.18A). Real-time PCR analysis revealed higher expression of ZEB1 andFOXC2 in ERG expressing cells (FIG. 18B). Data is first normalized for18S RNA and then expressed as fold induction over empty vectortransfected cells.

YK-4-279 Inhibits Expression of ERG Dependent EMT Markers

A549 cells express relatively high levels of ERG protein (FIG. 17). Weevaluated the EMT marker gene expression in these cells by real-timequantitative PCR (FIG. 19). TGF-β, a known EMT inducer, treatment ofA549 cells resulted in increased ZEB1 and FOXC2 expression. When theuntransfected A549 cells were treated with the ERG inhibitor, YK-4-279,we observed the complete opposite, both ZEB and FOXC2 expression wasinhibited.

The preliminary data presented here confirms that YK-4-279 directlybinds to ERG protein and inhibit its function as a transcription factor.Furthermore, we demonstrated that ERG can induce EMT markers in NSCLCcells and this effect can be reversed by YK-4-279.

Experiments summarized in this section test the hypothesis that ERGcontributes to the pathogenesis of NSCLC by inducing (EMT), and that ERGcan serve as a target for lung cancer therapy. We establish that ERG maybe successfully inhibited by YK-4-279 as a novel therapeutic approach.

The Role of ERG in Mediating EMT in Lung Cancer Cells

We assess the effects of modulating the levels of ERG expression on lungcancer cell lines, particularly regarding EMT. The H358 cell line,derived from a non-small cell lung cancer, expresses relatively lowlevels of endogenous ERG protein (FIG. 17). We introduce anERG-expression vector to elevate ERG levels in these cells. Other lungcancer cell lines (A549, H1944, H1395, and H596) express very highlevels of endogenous ERG. We use RNA interference techniques (shRNA orsiRNA) targeted to ERG to reduce its expression levels in these cells.In each case, the effect on EMT is assessed by determining theexpression levels of mRNAs and their cognate proteins that serve asspecific markers for EMT as follows: E-cadherin, vimentin, Snail, Slug,and ZEB1. A heightened EMT expression profile in H358 cellsoverexpressing ERG or a reduced EMT expression profile in cells (A549,H1944, H1395, and H596) in which ERG expression has been inhibited byRNA interference is observed, we extend these studies by determining theeffects of ZEB1 and ZEB 2 siRNAs. The hypothesis that ERG mediates EMTfunctions through ZEB1 and ZEB2 is supported if the effect of ERG on EMTis diminished when ZEB1 and ZEB2 expression is inhibited.

We determine IC50 values for common chemotherapeutic agents, cisplatin,paclitaxel, gemcitabine, etoposide, and vinblastine on five NSCLC celllines. Cell viability is determined by electric impedance and WSTassays. Once the baseline IC50 values are established, we repeat theexperiment with altered EGR expression. ERG expression is inhibited inA549, H1944, H1395, and H596 cells with shRNA. If stable shRNAexpression and reduced ERG protein expression cannot be achieved, wewill perform these experiments with transient transfection of siRNAtargeting ERG. Reducing ERG expression in NSCLC cell lines is expectedto shift the IC50 curves significantly to the left such that the cellsbecome more sensitive these chemotherapeutic agents. To complement theseexperiments we establish a stable H358 cell line that express highlevels of ERG protein from a mammalian expression vector. In this cellline we see a significant shift to right in the IC50 curve such that thecells become more resistant to chemotherapy.

New formulations of YK-4-279 that can be administered parenterally areproduced and the effects of YK-4-279 on the proliferation and malignantproperties of lung cancer cells are determined.

The lead excipient is β-hydroxypropyl cyclodextrin (β-HPCD), whileβ-HPCD is a clinically viable vehicle. The top seven formulations arecompared to kinetics for HPβCD. CD-1 are injected IP followed bytime-points at 0, 5, 10, 15, 30, 60, 120, 180, 240 and 480 minutes. A 24hour point also checks for delayed clearance. A series of CD-1 mice withIV injection followed by time-points at 0, 5, 10, 15, 30, 60, 120, 180,240 are used to measure absorption levels. Plasma are analyzed andpharmacokinetic parameters calculated. The goal of these studies is todetermine if there is a superior preparation to HPβCD by comparingabsorption and half-life. If a formulation can achieve IP absorption andsustain plasma levels of greater than 3 μM for 24 hours, we considerthis a significant improvement. This allows us to evaluate daily dosingin comparison with continuous infusion therapy. The use of a daily doserather than continuous IV is preferred for future animal and clinicalstudies.

We inhibit ERG function by treating cells with YK-4-279. Changes in EMTmarkers are determined as described above to confirm that YK-4-279treatment results in the same EMT marker expression profile as the lackof ERG (siRNA or shRNA). The goal of following experiments is to assessthe effects on functional outcomes when EMT is altered. For thispurpose, we evaluate surrogate markers of the malignant phenotype asfollows: cell motility, chemotaxis, invasion of an endothelial cellmonolayer, growth on plastic, growth in soft agar, and in vivo growth asxenografts. In parallel experiments, cells are treated with YK-4-279 andvarying concentrations of different chemotherapeutic agents for NSCLC,including cisplatin, paclitaxel, gemcitabine, etoposide, andvinblastine. We observe a synergistic inhibitory effect induced byYK-4-279 in combination with these drugs. To support the hypotheses thatinhibiting ERG expression diminishes EMT through ZEB1/2, we determinewhether enforced overexpression of ZEB1/2 reverses the effects ofYK-4-279 on cell phenotype.

We test the effects of YK-4-279 on NSCLC cell motility and invasion. Wetest YK-4-279 for its ability to inhibit NSCLC cell invasion using thexCELLigence system. This new method allows real-time measurement of cellmotility in a classical Boyden chamber format with a layer of goldelectrodes on the underneath surface of the porous membrane (xCELLigencesim-plates). As the cells move from the upper chamber through themembrane towards a chemoattractant in the lower chamber, they increaseelectric impedance on the under surface of the membrane, which isrecorded in real-time. The same instrument is also used for measuringinvasion through an endothelial monolayer (Rahim S, Uren A. A real-timeelectrical impedance based technique to measure invasion of endothelialcell monolayer by cancer cells. Journal of visualized experiments: JoVE.2011). In this experimental format, human umblical vein endothelialcells (HUVEC) grow on regular cell culture plates with gold electrodeson the surface (xCELLigence E-plates). Once the endothelial cells form astable monolayer, NSCLC cells are added on top. As the cancer cellsbreak tight junctions between endothelial cells and penetrate throughthe endothelial monolayer, they alter the electric impedance. Theseexperiments allow us to evaluate if YK-4-279 alters motility, chemotaxisand invasive phenotype of NSCLC cells

We perform synergy studies in cell culture by titrating YK-4-279 andchemotherapeutic agents (cisplatin, paclitaxel, gemcitabine, etoposide,and vinblastine). Cell death is used as the end point and any potentialsynergy is calculated by combination index (CI) isobologram equationmethod (Chou T C. Theoretical basis, experimental design, andcomputerized simulation of synergism and antagonism in drug combinationstudies. Pharmacological reviews. 2006; 58:621-81).

We test the effect of ERG inhibition on growth of 3 different humanNSCLC xenografts (two with high ERG expression and one with low ERGexpression). Cell suspension prepared in matrigel is subcutaneouslyimplanted into four- to six-week-old male SCID mice. Adjusting for takerate, two groups of animals (10 animals/xenograft line) are administeredthe inhibitor YK-4-279, and the carrier only placebo based on theformulation studies for each xenograft line. Drug treatment start whenthe tumors reach to 200 mm3 size. Tumor growth and body weight ismeasured twice weekly. All experimental groups have a power of 83% withp<0.05 to detect a 35% difference in total tumor volume. The animals areharvested at eight weeks or earlier if animals become compromised(primary tumor reaching to 2000 mm³, primary tumor ulcerating, or miceshowing signs of pain and distress). Half of the tumor tissue isembedded in paraffin for immunohistochemical analysis and the other halfflash frozen for molecular analysis. We hypothesize that blocking ERGactivity in NSCLC xenografts may result in a reduction in primary tumorsize.

While the disclosure has been illustrated and described in detail in thedrawings and foregoing description, such illustration and descriptionare to be considered illustrative or exemplary and not restrictive. Thedisclosure is not limited to the disclosed embodiments. Variations tothe disclosed embodiments can be understood and effected by thoseskilled in the art in practicing the claimed disclosure, from a study ofthe drawings, the disclosure and the appended claims.

All references cited herein are incorporated herein by reference intheir entirety. To the extent publications and patents or patentapplications incorporated by reference contradict the disclosurecontained in the specification, the specification is intended tosupersede and/or take precedence over any such contradictory material.

Unless otherwise defined, all terms (including technical and scientificterms) are to be given their ordinary and customary meaning to a personof ordinary skill in the art, and are not to be limited to a special orcustomized meaning unless expressly so defined herein. It should benoted that the use of particular terminology when describing certainfeatures or aspects of the disclosure should not be taken to imply thatthe terminology is being re-defined herein to be restricted to includeany specific characteristics of the features or aspects of thedisclosure with which that terminology is associated. Terms and phrasesused in this application, and variations thereof, especially in theappended claims, unless otherwise expressly stated, should be construedas open ended as opposed to limiting. As examples of the foregoing, theterm ‘including’ should be read to mean ‘including, without limitation,’‘including but not limited to,’ or the like; the term ‘comprising’ asused herein is synonymous with ‘including,’ ‘containing,’ or‘characterized by,’ and is inclusive or open-ended and does not excludeadditional, unrecited elements or method steps; the term ‘having’ shouldbe interpreted as ‘having at least;’ the term ‘includes’ should beinterpreted as ‘includes but is not limited to;’ the term ‘example’ isused to provide exemplary instances of the item in discussion, not anexhaustive or limiting list thereof; adjectives such as ‘known’,‘normal’, ‘standard’, and terms of similar meaning should not beconstrued as limiting the item described to a given time period or to anitem available as of a given time, but instead should be read toencompass known, normal, or standard technologies that may be availableor known now or at any time in the future; and use of terms like‘preferably,’ ‘preferred,’ desired,′ or ‘desirable,’ and words ofsimilar meaning should not be understood as implying that certainfeatures are critical, essential, or even important to the structure orfunction of the invention, but instead as merely intended to highlightalternative or additional features that may or may not be utilized in aparticular embodiment of the invention. Likewise, a group of itemslinked with the conjunction ‘and’ should not be read as requiring thateach and every one of those items be present in the grouping, but rathershould be read as ‘and/or’ unless expressly stated otherwise. Similarly,a group of items linked with the conjunction ‘or’ should not be read asrequiring mutual exclusivity among that group, but rather should be readas ‘and/or’ unless expressly stated otherwise.

Where a range of values is provided, it is understood that the upper andlower limit, and each intervening value between the upper and lowerlimit of the range is encompassed within the embodiments.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity. The indefinite article “a” or “an” does not exclude aplurality. A single processor or other unit may fulfill the functions ofseveral items recited in the claims. The mere fact that certain measuresare recited in mutually different dependent claims does not indicatethat a combination of these measures cannot be used to advantage. Anyreference signs in the claims should not be construed as limiting thescope.

It will be further understood by those within the art that if a specificnumber of an introduced claim recitation is intended, such an intentwill be explicitly recited in the claim, and in the absence of suchrecitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should typically be interpreted to mean “atleast one” or “one or more”); the same holds true for the use ofdefinite articles used to introduce claim recitations. In addition, evenif a specific number of an introduced claim recitation is explicitlyrecited, those skilled in the art will recognize that such recitationshould typically be interpreted to mean at least the recited number(e.g., the bare recitation of “two recitations,” without othermodifiers, typically means at least two recitations, or two or morerecitations). Furthermore, in those instances where a conventionanalogous to “at least one of A, B, and C, etc.” is used, in generalsuch a construction is intended in the sense one having skill in the artwould understand the convention (e.g., “a system having at least one ofA, B, and C” would include but not be limited to systems that have Aalone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). In those instances where aconvention analogous to “at least one of A, B, or C, etc.” is used, ingeneral such a construction is intended in the sense one having skill inthe art would understand the convention (e.g., “a system having at leastone of A, B, or C” would include but not be limited to systems that haveA alone, B alone, C alone, A and B together, A and C together, B and Ctogether, and/or A, B, and C together, etc.). It will be furtherunderstood by those within the art that virtually any disjunctive wordand/or phrase presenting two or more alternative terms, whether in thedescription, claims, or drawings, should be understood to contemplatethe possibilities of including one of the terms, either of the terms, orboth terms. For example, the phrase “A or B” will be understood toinclude the possibilities of “A” or “B” or “A and B.”

All numbers expressing quantities of ingredients, reaction conditions,and so forth used in the specification are to be understood as beingmodified in all instances by the term ‘about.’ Accordingly, unlessindicated to the contrary, the numerical parameters set forth herein areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of anyclaims in any application claiming priority to the present application,each numerical parameter should be construed in light of the number ofsignificant digits and ordinary rounding approaches.

Furthermore, although the foregoing has been described in some detail byway of illustrations and examples for purposes of clarity andunderstanding, it is apparent to those skilled in the art that certainchanges and modifications may be practiced. Therefore, the descriptionand examples should not be construed as limiting the scope of theinvention to the specific embodiments and examples described herein, butrather to also cover all modification and alternatives coming with thetrue scope and spirit of the invention.

What is claimed is:
 1. A method of treating a subject having a cancerselected from the group consisting of lung cancer and glioblastomamultiforme, the method comprising administering to a subject in needthereof an effective amount of a compound having the structure:

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the lung cancer comprises a non-small-cell lung carcinoma. 3.The method of claim 1, wherein the lung cancer comprises a glioblastomamultiforme.
 4. The method of claim 1, wherein the cancer comprises atranslocation comprising an ETS gene selected from the group consistingof FLI1, ETV1, ETV4, ERG, ETS1, and ETS2.
 5. The method of claim 1,wherein the compound is administered in combination with an additionalchemotherapeutic agent.
 6. The method of claim 1, wherein the additionalchemotherapeutic agent is selected from the group consisting ofcisplatin, paclitaxel, gemcitabine, etoposide, and vinblastine.
 7. Themethod of claim 1, wherein the compound is administered parentally. 8.The method of claim 1, wherein the compound substantially consists ofthe (S) enantiomer.
 9. The method of claim 1, wherein the compound isadministered in combination with a β-hydroxypropyl cyclodextrinexcipient.
 10. The method of claim 1, wherein the subject is mammalian.11. The method of claim 1, wherein the subject is human.
 12. A method ofinhibiting the growth of a neoplastic cell selected from the groupconsisting of a lung cell and a glial cell, the method comprisingcontacting the cell with a compound having the structure:


13. The method of claim 12, wherein the cell is a glioblastomamultiforme cell.
 14. The method of claim 12, wherein the cell is aglioblastoma cell selected from the group consisting of a DKMG cell, aDBTRG cell, a 42MGBA cell, a GAMG cell, a U87MG cell, a H4 cell, and a8MGBA cell.
 15. The method of claim 12, wherein the cell is anon-small-cell lung carcinoma cell.
 16. The method of claim 12, whereinthe cell is a lung cell selected from the group consisting of a A549cell, a H1944 cell, a H358 cell, a H1395 cell, and a H596 cell.
 17. Themethod of claim 12, wherein the cell is in vivo.
 18. The method of claim12, wherein the cell is in vitro.
 19. The method of claim 12, whereinthe subject is mammalian.
 20. The method of claim 12, wherein thesubject is human.